JP2017512752A - Hydrophilic antibody-drug conjugate - Google Patents

Abstract

Provided are hydrophilic linkers, drug-linker compounds, drug-ligand conjugate compounds and ligand-linkers, and methods of making and using them. The present invention provides, inter alia, hydrophilic ligand-linker-drug conjugates. By designing conjugates that have similar hydrophilicity to unconjugated targeting agents (eg, antibodies), the conjugates are similar to the pharmacokinetic properties of unconjugated targeting agents in vivo. Retains the ability to provide pharmacokinetic properties.

Description

  This application takes advantage of US Provisional Patent Application No. 61 / 940,759, filed February 17, 2014, and US Provisional Patent Application No. 61 / 947,368, filed March 3, 2014. Insist. Each of these US provisional patent applications is incorporated herein in its entirety.

BACKGROUND OF THE INVENTION There has been great interest in the use of monoclonal antibodies (mAbs) for targeted delivery of cytotoxic agents to cancer cells. The design of antibody drug conjugates, typically by linking a cytotoxic agent to an antibody via a linker involves considering various factors. These factors include the identity and location of the chemical group (s) for conjugation of the cytotoxic agent, the mechanism of release of the cytotoxic agent, and the structural element (s) that provide the release of the agent ( As well as structural modifications of the released release agent (if present). Furthermore, if the cytotoxic agent is released after internalization of the antibody, the structural elements for drug release and the mechanism of drug release must be consistent with the intracellular transport of the conjugate.

  While several different drug classes have been evaluated for antibody-mediated delivery, only a few drug classes have adequate toxicity profiles while sufficient to authorize clinical development as antibody-drug conjugates. Has been proven to be active. One such class is auristatin associated with the natural product dolastatin 10. Exemplary auristatins include MMAE (N-methylvaline-valine-dolaproine-dolaproine-norephedrine) and MMAF (N-methylvaline-valine-dolaproine-dolaproine-phenylalanine).

MMAE is an example of a cytotoxic agent that is active as a free drug, is highly potent after conjugation to a monoclonal antibody (mAb), and is released after internalization into the cell. MMAE is a peptide based linker that can be cleaved by cathepsin B containing maleimidocaproyl-valine-citrulline (mc-vc-) and the self-destroying group p-aminobenzyl-carbamoyl (PABC). Successfully conjugated to a mAb at the terminal amino acid, an antibody-drug conjugate of the following structure, mAb- (mc-vc-PABC-MMAE) _p has been produced. (In the previous formula, p refers to the number of (mc-vc-PABC-MMAE) units per mAb or antibody.) By breaking the bond between the vc peptide and the self-destroying PABC group, The group itself is released from MMAE, liberating free MMAE.

Another auristatin, MMAF, is less active as a free drug (compared to MMAE) but is highly potent after conjugation to antibodies, internalization into cells and release. MMAF is linked to MMAF N via a peptide-based linker cleavable by cathepsin B containing maleimidocaproyl-valine-citrulline (mc-vc-) and the self-destroying group p-aminobenzyl-carbamoyl (PABC). Successfully conjugated to a monoclonal antibody (mAb) at the terminal amino acid, the structure, mAb- (mc-vc-PABC-MMAF) _p (p is mAb or (mc-vc-PABC-MMAF) units per antibody) Antibody-drug conjugates have been produced. By breaking the bond between the peptide and the PABC subunit, the self-destructing PABC group releases the self-destructing PABC group itself from the MMAF, releasing the free MMAF.

MMAF is also active as a non-cleavable conjugate containing maleimidocaproyl MMAF (mcMMAF), a drug-linker. When this conjugate, mAb- (mcMMAF) _p, is internalized into the cell, the active species released is cys-mcMMAF. Since the linker is not cleavable, the maleimidocaproyl and antibody cysteine residues remain attached to the N-terminus of MMAF. MMAF was also reported to be active as a C-terminal conjugate attached to the peptide-maleimidocaproyl linker at its C-terminal amino acid phenylalanine. When this conjugate (MMAF-peptide-mc) _p -mAb is internalized into the cell, the active species MMAF is released following cleavage of the MMAF (phenylalanine) -peptide bond.

  In animal models, these MMAE and MMAF conjugates generally showed a drug load dependent decrease in pharmacokinetic properties. In particular, as the number of drug-linker units attached to each antibody increased, the pharmacokinetics (PK) of the conjugate decreased.

  Thus, another important factor in the design of antibody-drug conjugates is the amount of drug that can be delivered per targeting agent (ie, cells that are bound to each targeting agent (eg, antibody). The number of toxic agents, referred to as drug load or drug load). Historically, there has been a hypothesis that higher drug loads are superior to lower drug loads (eg, 8 loads versus 4 loads). There was a theoretical interpretation that the higher loaded conjugate delivered more drug (cytotoxic agent) to the targeted cells. This theoretical interpretation was supported by the observation that conjugates with higher drug loadings were more active against cell lines in vitro. However, certain subsequent studies revealed that this hypothesis was not confirmed in animal models. Conjugates with 4 or 8 drug loads of certain auristatins were observed to have similar activity in a mouse model. For example, Hamblett et al., Clinical Cancer Res. 10: 7063-70 (2004). Further reported that higher loaded ADCs were cleared more rapidly from circulation in animal models. This faster clearance suggested a trend for PK for the higher loaded species compared to the lower loaded species. For example, Hamlett et al. Furthermore, the higher loaded conjugate had a lower MTD in the mice, resulting in a narrower therapeutic index reported. Same as above. In contrast, an ADC with a drug load of 2 at the engineered site of a monoclonal antibody has been reported to have the same or better PK properties and therapeutic index compared to a particular 4-loaded ADC. It was. For example, Junutula et al., Clinical Cancer Res. 16: 4769 (2010). Therefore, a recent trend is to develop ADCs with low drug loading.

An alternative approach to overcome the PK tendency of higher loaded ADCs was to attach solubilizing groups to the ADC. For example, polyethylene glycol polymers or other water soluble polymers have been included in linkers (eg, between drug and antibody binding sites) in an attempt to overcome the tendency of PK. Another approach was to attach a drug-polymer to the antibody, but each polymer contains a number of drugs. However, these alternatives did not necessarily achieve the desired results. Furthermore, attaching solubilizing groups can increase the complexity of manufacturing such conjugates.
Thus, other desirable characteristics of lower loaded conjugates, such as antibody drug conjugate formats (and more generally other conjugates) that allow higher drug loading while maintaining favorable PK properties. There is still a need for a format for gates. Surprisingly, the present invention addresses these needs.

Hamlett et al., Clinical Cancer Res. 2004, volume 10, pp. 7063-70 Junutula et al., Clinical Cancer Res. 2010, Vol. 16, p. 4769

BRIEF SUMMARY OF THE INVENTION The present invention provides, inter alia, hydrophilic ligand-linker-drug conjugates. By designing a conjugate that has similar hydrophilicity to an unconjugated targeting agent (eg, a ligand, eg, an antibody), the conjugate is an unconjugated targeting agent drug in vivo. Retains the ability to provide pharmacokinetic (PK) properties similar to kinetic (PK) properties. Conjugates also retain such desirable PK properties and have the same or better activity in vivo while higher drug loading (ie, target) compared to lower loaded conjugates. There can be a greater number of hydrophilic drugs-linkers) per agent. (For example, 4 loaded or 8 loaded conjugates can have the same or better PK characteristics than their 2 loaded or 4 loaded counterparts. Such 4 loaded or 8 loaded The conjugates can have the same or better activity as their 2-loaded or 4-loaded counterparts, respectively). Thus, a targeting agent selected based on certain desired properties can be conjugated with a drug linker without substantially compromising the desired properties, such as the PK properties of the targeting agent alone. Also provided are methods of making and using such conjugates.

  Also provided are linker drug compounds for conjugation to targeting agents (ligands) and methods of preparing and using such compounds. Further provided are ligand-linker compounds to which drug compounds can be attached, as well as methods of preparing and using such compounds.

FIG. 1 shows an exemplary synthetic scheme for the assembly of ligand-linker-drug conjugates described herein.

FIG. 2 shows the results of a mouse study comparing the pharmacokinetic stability of unconjugated antibody, two hydrophilic ADCs and three control ADCs. The ADCs are shown in order from top to bottom.

FIG. 3 shows the results of a mouse study comparing the pharmacokinetic stability of five hydrophilic ADCs and a control ADC.

FIG. 4 shows the results of HIC chromatography of the antibody drug conjugate.

FIG. 5 shows the results of HIC chromatography of the antibody drug conjugate.

FIG. 6 shows the results of a mouse xenograft study comparing the activity of 4-loaded and 8-loaded ADCs.

FIG. 7 shows the results of a mouse xenograft study comparing the activity of 4-loaded and 8-loaded ADCs.

FIG. 8 shows the results of a mouse xenograft study comparing the activity of 4- and 8-loaded ADCs.

FIG. 9 shows the results of a mouse xenograft study comparing the activity of 4- and 8-loaded ADCs.

Abbreviations and Definitions Unless otherwise noted, the following terms and phrases, as used herein, are intended to have the following meanings. As trade names are used herein, trade names include product formulations, generic drugs, and the active pharmaceutical ingredient (s) of a branded product, unless otherwise indicated by context.

  The term “hydrophilic index” refers to a measure of the hydrophilicity of a conjugate relative to the hydrophilicity of a targeting agent alone (ie, a ligand, typically an antibody). The hydrophilicity index is the corresponding unconjugated targeting agent (single) under high performance liquid chromatography (HPLC) conditions as the retention time of a conjugate, as further described herein. Measured against the retention time. For example, the retention time of a ligand-linker-drug conjugate relative to the retention time of an unconjugated ligand (typically an antibody) can be determined. In selected embodiments, the retention time of the conjugate will not be delayed more than 2 minutes from the retention time of the unconjugated ligand as determined in the examples (hydrophilicity index of 2). Called). In certain embodiments, the retention time of the conjugate does not lag more than 1 minute from the retention time of the unconjugated ligand as determined in the examples (1 hydrophilicity). Called the index). In certain embodiments, the retention time of the conjugate is not delayed more than 0.5 minutes from the retention time of the unconjugated ligand, as determined in the Examples (0. Referred to as a hydrophilic index of 5). If a different hydrophobic interaction column and / or method is used, this is calibrated using the conjugate from Table 2 as a reference, and the mobility of the reference conjugate on the selected column and / or method. (Elution time) can be determined. The selected hydrophobic interaction column and / or the determined reference mobility on the method can then be used to calculate the hydrophilicity index of the test substance (as determined according to Example 3). . For example, auristatin T-Glu-Dpr-MA, mc-MMAF and mc-vc-PABC-MMAE drug linker can be used to form a conjugate for use as a reference. In another example, auristatin T-Glu-Dpr-MA-h1F6 ADC, h1F6-mc-MMAF and h1F6-mc-vc-PABC-MMAE ADC can be used as a reference.

The term “alkyl” by itself or as part of another substituent, unless otherwise indicated, means a straight or branched hydrocarbon radical having the indicated number of carbon atoms (ie, C _{1 ~ 8} means 1-8 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like. . The term “alkenyl” refers to an unsaturated alkyl group having one or more double bonds. Similarly, the term “alkynyl” refers to an unsaturated alkyl group having one or more triple bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3- (1,4-pentadienyl), ethynyl, 1 -And 3-propynyl, 3-butynyl, and higher homologs and isomers are included. The term “cycloalkyl” has the indicated number of ring atoms (eg, C _3-6 cycloalkyl), is fully saturated, or has no more than one double bond between the vertices of the ring. Refers to the hydrogen ring. “Cycloalkyl” is also intended to refer to bicyclic and polycyclic hydrocarbon rings such as bicyclo [2.2.1] heptane, bicyclo [2.2.2] octane, and the like. The term “heterocycloalkane” or “heterocycloalkyl” refers to a cycloalkyl group containing 1 to 5 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized. And the nitrogen atom (s) are optionally quaternized. The heterocycloalkane may be a monocyclic ring system, a bicyclic ring system, or a polycyclic ring system. Non-limiting examples of heterocycloalkane groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine- S-oxide, thiomorpholine-S, S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine and the like are included. A heterocycloalkane group can be attached to the remainder of the molecule through a ring carbon or a heteroatom.

The term “alkylene” by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified by —CH ₂ CH ₂ CH ₂ CH ₂ —. Typically, an alkyl (or alkylene) group has 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having four or fewer carbon atoms. Similarly, “alkenylene” and “alkynylene” refer to unsaturated forms of “alkylene” having double or triple bonds, respectively.

As used herein, a wavy line that intersects a single, double, or triple bond in any chemical structure shown herein
Represents the point of attachment of a single, double or triple bond to the rest of the molecule.

The terms “alkoxy”, “alkylamino” and “alkylthio” (or thioalkoxy) are used in their usual sense and are attached to the rest of the molecule through an oxygen, amino or sulfur atom, respectively. Refers to an alkyl group. Furthermore, for dialkylamino groups, the alkyl moieties may be the same or different and together can form a 3-7 membered ring with the nitrogen atom to which each is attached. Thus, a group represented as dialkylamino or —NR ^a R ^b is intended to include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl and the like.

The term “halo” or “halogen” by itself or as part of another substituent means a fluorine, chlorine, bromine, or iodine atom unless otherwise specified. Furthermore, the term, for example, “haloalkyl” is intended to include monohaloalkyl and polyhaloalkyl. For example, the term “C _1-4 haloalkyl” is intended to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

  The term “aryl”, unless stated otherwise, means a polyunsaturated, typically aromatic hydrocarbon group, which may be a single ring, fused to one, or It may be a plurality of rings (up to 3 rings) that are covalently linked. The term “heteroaryl” refers to an aryl group (or ring) containing 1 to 5 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, The nitrogen atom (s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl groups include phenyl, naphthyl and biphenyl, while non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, Phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzoisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, Benzothiazolyl, benzofuranyl, benzothienyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyra Including Lil, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl and the like. The substituents for each of the aryl and heteroaryl ring systems noted above are selected from the group of permissible substituents below when described as being “substituted”.

  The term “arylalkyl” is intended to include radicals in which an aryl group is attached to an alkyl group (eg, benzyl, phenethyl, etc.). Similarly, the term “heteroaryl-alkyl” is intended to include radicals in which a heteroaryl group is attached to an alkyl group (eg, pyridylmethyl, thiazolylethyl, etc.).

  The above terms (eg, “alkyl”, “aryl” and “heteroaryl”) include, in some embodiments, both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

Unless otherwise indicated by the context, substituents for alkyl radicals (including groups often referred to as alkylene, alkenyl, alkynyl and cycloalkyl) range from 0 to (2m ′ + 1), where m ′ is -Halogen, -OR ', -NR'R ", -SR', -SiR'R" R '''in a number ranging up to the total number of carbon atoms in such radicals) , —OC (O) R ′, —C (O) R ′, —CO ₂ R ′, —CONR′R ″, —OC (O) NR′R ″, —NR ″ C (O) R ', -NR'-C (O) NR''R''', - NR''C (O) 2 R ', - NH-C (NH 2) = NH, -NR'C (NH 2) = NH, —NH—C (NH ₂ ) ═NR ′, —S (O) R ′, —S (O) ₂ R ′, —S (O) ₂ NR′R ″, —NR ′S (O) Species selected from ₂ R ″, —CN and —NO ₂ Various groups are acceptable. R ′, R ″ and R ′ ″ are each independently hydrogen, unsubstituted C _1-8 alkyl, unsubstituted aryl, aryl substituted with 1 to 3 halogens, unsubstituted C _1-8 Refers to an alkyl, C _1-8 alkoxy or C _1-8 thioalkoxy group, or an unsubstituted aryl-C _1-4 alkyl group. When R ′ and R ″ are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, —NR′R ″ is intended to include 1-pyrrolidinyl and 4-morpholinyl.

Similarly, the substituents for aryl and heteroaryl groups vary and include —halogen, —OR ′, —OC (O), ranging from 0 to the total number of open valences on the aromatic ring system. R ′, —NR′R ″, —SR ′, —R ′, —CN, —NO ₂ , —CO ₂ R ′, —CONR′R ″, —C (O) R ′, —OC (O ) NR′R ″, —NR ″ C (O) R ′, —NR ″ C (O) ₂ R ′, —NR′—C (O) NR ″ R ′ ″, —NH—C (NH ₂ ) ═NH, —NR′C (NH ₂ ) ═NH, —NH—C (NH ₂ ) ═NR ′, —S (O) R ′, —S (O) ₂ R ′, —S ( Generally selected from O) ₂ NR′R ″, —NR ′S (O) ₂ R ″, —N ₃ , perfluoro (C ₁ -C ₄ ) alkoxy, and perfluoro (C ₁ -C ₄ ) alkyl; R ', R''andR''' is _{hydrogen, C 1 to 8} alkyl C _{1 to 8 haloalkyl, C 3 to 6 cycloalkyl, C 2 to 8 alkenyl, C 2 to 8} alkynyl, unsubstituted aryl and heteroaryl, (unsubstituted _{aryl) -C 1 to 4} alkyl, and unsubstituted aryloxy - Independently selected from C _1-4 alkyl. Other suitable substituents include each of the above aryl substituents attached to a ring atom by an alkylene tether of 1 to 4 carbon atoms.

The term “base” refers to a functional group that deprotonates water to produce hydroxide ions. Exemplary bases are amines and nitrogen-containing heterocycles. Exemplary bases include —N (R ³ ) (R ⁴ ), where R ³ and R ⁴ are H or C _1-6 alkyl, preferably H or methyl,
Wherein R ⁵ , R ⁶ , R ⁷ and R ⁸ are independently selected each time from hydrogen or C _1-6 alkyl, preferably H or methyl, and e is 0 ~ 4. In some embodiments, the base is a nitrogen base.

  The term “antibody” is used herein in the broadest sense and is an intact monoclonal antibody, polyclonal antibody, monospecific antibody, multispecific antibody (eg, bispecific antibody), and the desired organism. Specifically covered are antibody fragments that exhibit activity (ie, specific binding to a target antigen). An intact antibody has two main regions: a variable region and a constant region. The variable region specifically binds to and interacts with the target antigen. The variable region includes a complementarity determining region (CDR) that recognizes and binds to a specific binding site on a particular antigen. The constant region is recognized by and can interact with the immune system (see, eg, January et al., 2001, Immuno. Biology, 5th edition, Garland Publishing, New York). The antibodies can be of any type (eg, IgG, IgE, IgM, IgD, and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. The antibody can be derived from any suitable species. In some embodiments, the antibody is of human or mouse origin. A monoclonal antibody can be, for example, human, humanized or chimeric.

  The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population can be present in trace amounts. Except for mutations present in Monoclonal antibodies are highly specific and are directed to a single antigenic site. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

An “intact antibody” is one that includes the antigen binding variable region and the light chain constant domain (C _L ) and heavy chain constant domains CH ₁ , CH ₂ , CH ₃ and CH ₄ as appropriate for the antibody class. . The constant domain can be a native sequence constant domain (eg, a human native sequence constant domain) or an amino acid sequence variant thereof.

“Antibody fragments” comprise a portion of an intact antibody, said portion comprising its antigen binding or variable region. Examples of antibody fragments include Fab, Fab ′, F (ab ′) ₂ , and Fv fragments, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, linear antibodies, single chain antibody molecules, scFv, scFv Fc, multispecific antibody fragment formed from antibody fragment (s), fragment (s) produced by Fab expression library, or target antigen (eg cancer cell antigen, viral antigen or microbial antigen) Epitope binding fragments of any of the above that specifically bind to are included.

  An “antigen” is an entity to which an antibody specifically binds. The antigen can be, for example, proteinaceous (eg, protein, polypeptide or peptide), non-proteinaceous (eg, carbohydrate), or a combination of the two.

The terms “specific binding” and “specifically bind” refer to a targeting agent or ligand, eg, an antibody or antigen-binding fragment, that binds its corresponding target antigen in a highly selective manner, and numerous other Does not bind to any antigen. For antibodies, the antibody or antibody fragment is typically at least about 1 × 10 ⁻⁷ M, preferably 10 ⁻⁸ M to 10 ⁻⁹ M, 10 ⁻¹⁰ M, 10 ⁻¹¹ M, or 10 ⁻¹² M. Bind with affinity and with an affinity that is at least 2 times greater than its affinity for binding to a given antigen with the same epitope and a non-specific antigen other than a closely related antigen (eg, BSA, casein) To bind to a predetermined antigen.

  The term “inhibit” or “inhibition” means to reduce or totally prevent by a measurable amount.

  The term “therapeutically effective amount” refers to an amount of a conjugate (eg, antibody drug conjugate) effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of the conjugate reduces the number of cancer cells; reduces the size of the tumor; inhibits the invasion of cancer cells into peripheral organs (ie slows to some extent, preferably May inhibit tumor metastasis (ie slow, preferably stop) to some extent; inhibit tumor growth; and / or alleviate one or more of the symptoms associated with cancer. To the extent that the drug can inhibit growth and / or kill existing cancer cells, the drug can be cytostatic and / or cytotoxic. For cancer therapy, efficacy can be measured, for example, by assessing time to disease progression (TTP) and / or determining response rate (RR).

  The term “substantial” or “substantially” means the majority of the mixture or sample of the population, ie> 50%, preferably more than 50%, 55%, 60%, 65%, Over 70%, over 75%, over 80%, over 85%, over 90%, over 91%, over 92%, over 93%, over 94%, over 95%, over 96%, over 97%, over 98% Over, or over 99%.

The terms “intracellularly cleaved” and “intracellularly cleaved” refer to intracellular metabolic processes or reactions on a ligand-linker-drug conjugate (eg, antibody drug conjugate (ADC), etc.), thereby The covalent bond (linker unit) between the _DE moiety and the ligand unit (eg, antibody (Ab)) is broken, resulting in the release of the _DE unit. Thus, the cleaved portion of the ligand-linker-drug conjugate is an intracellular metabolite.

The term “cytotoxic activity” refers to the cell killing or cytotoxic effect of a ligand-linker-drug conjugate compound, typically via a released drug unit on a target cell. Cytotoxic activity can be expressed as an IC ₅₀ value (also referred to as half-maximal inhibitory concentration), which is the concentration (molar or mass) per unit volume at which half of the cells survive from exposure to the conjugate.

  The term “cytotoxic agent” as used herein refers to a substance that kills cells or otherwise causes destruction of cells.

  The terms “cancer” and “cancerous” refer to or describe the physiological condition or disorder in mammals that is typically characterized by unregulated cell growth. “Tumor” includes cancerous cells.

  An “autoimmune disease” is a disease or disorder that arises from and against an individual's own tissues or proteins.

  Examples of “patients” include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds and poultry. In an exemplary embodiment, the patient is a human.

  The terms “treat” or “treatment” refer to therapeutic treatment and preventative measures to prevent recurrence, unless indicated otherwise by context, the purpose of which is to treat unwanted physiological changes or disorders such as To inhibit or slow (reduce) the development or spread of cancer. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, attenuated degree of disease, stabilized (ie, not exacerbated) condition of disease, whether detectable or non-detectable. Includes delay or slowing of disease progression, recovery or alleviation of disease state, and remission (whether partial or complete). “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder.

  In the context of cancer, the term “treating” inhibits tumor cell, cancer cell, or tumor growth, inhibits tumor cell or cancer cell replication, and reduces overall tumor burden. Or reducing the number of cancerous cells and ameliorating one or more symptoms associated with the disease.

  In the context of an autoimmune disease, the term “treat” includes, but is not limited to, inhibiting cell replication associated with an autoimmune disease state, including cells that produce autoimmune antibodies, autoimmunity Any or all of reducing the amount of sex antibody and ameliorating one or more symptoms of an autoimmune disease.

  The phrase “pharmaceutically acceptable salt” as used herein refers to a pharmaceutically acceptable organic salt of a compound (eg, drug, drug-linker, or ligand-linker-drug conjugate) or Refers to inorganic salt. The compound can contain at least one amino group, and thus acid addition salts can be formed with amino groups. Exemplary salts include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, hydrogensulfate, phosphate, acidic phosphorus Acid salt, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, hydrogen tartrate, ascorbate, succinate, maleate, Gentisinate, fumarate, gluconate, glucuronate, saccharide, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfone Acid salts, and pamoates (ie, 1,1′-methylene-bis- (2-hydroxy-3-naphthoate)). A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as acetate ion, succinate ion or other counterion. The counter ion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt can have multiple charged atoms in its structure. When multiple charged atoms are part of a pharmaceutically acceptable salt, they can have multiple counter ions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterion.

DETAILED DESCRIPTION SUMMARY The present invention uses similar combinations of linking groups and cytotoxic agents to mimic the hydrophilicity of unconjugated ligands (ie targeting agents such as antibodies or antigen-binding fragments). Based in part on the discovery that ligand-linker-drug conjugates, eg, antibody-drug conjugates (ADC), having hydrophilicity can be prepared. By maintaining the hydrophilicity of the conjugate similar to the hydrophilicity of the unconjugated ligand, the conjugate thus obtained will have certain desirable characteristics of the ligand alone, such as reduced clearance in vivo. Higher drug loadings (eg, at least 4 or 8 drug linkers per ligand) while maintaining increased in vivo pharmacokinetic profile, increased exposure of the conjugate to the target cell (s), etc. ). Advantageously, such hydrophilic conjugates have a hydrophilicity similar to that of the ligand without the need to include additional solubilizing groups such as polyethylene glycol or other water soluble polymers. Can be designed as (Ligand-linker-drug conjugates are also referred to herein as drug-ligand conjugates, ligand-drug conjugates or ligand drug conjugates.)

The hydrophilic linking group of the conjugate (also referred to as a linker or linker unit) is designed for increased hydrophilicity. The linking group, in the case of cytotoxic agents, allows for efficient release of cytotoxic agents (also referred to as drug units or drugs) in the target cells sufficient to induce cytotoxic or cytostatic effects. . Typically, hydrophilic linkers are designed for efficient release of drug units immediately after the conjugate is internalized into the target cell. Appropriate recognition sites for the release of drug units by cleavage are those that allow efficient separation of the units from the hydrophilic linking group. Typically, the recognition site is a peptide cleavage site (eg, formed in combination with a cytotoxic agent having amino acid or peptide characteristics at the site of attachment to the linking group). Examples of peptide cleavage sites include those recognized by intracellular proteases, such as those present in lysosomes. In embodiments where the linking group is attached to the amino acid of the effector moiety (D _E ), AA ₁ forms a cleavable peptide bond with the D _E unit. A cleavable peptide bond is susceptible to cleavage by a protease when the conjugate reaches its targeted site. In other embodiments, AA ₁ forms an amide bond with the binding site of an effector moiety (D _E ) that is susceptible to cleavage (eg, by a protease) when the conjugate reaches its targeted site. .

  In some embodiments, the drug unit is auristatin designed to have increased hydrophilicity in combination with a hydrophilic linker unit. The drug unit can advantageously be designed to have hydrophilic substituents while retaining potent cytotoxic activity. The auristatin drug unit is attached to the linker unit at their C-terminus, as described more fully herein.

Thus, in some embodiments, L ^H is a hydrophilic linker containing 1, 2, 3 or 4 hydrophilic amino acids, wherein the first amino acid is the drug unit to which it is attached. A cleavage site is formed together with the part of. In some embodiments, L ^H is a hydrophilic linker comprising one or two hydrophilic amino acids, and the first amino acid forms a cleavage site with the portion of the drug unit to which it is attached. In some embodiments, L ^H is a hydrophilic linker comprising an amino acid that forms a cleavage site with the portion of the drug unit to which it is attached. In some embodiments, the second, third and / or fourth hydrophilic amino acid is replaced with an optionally substituted alkylene or heteroalkylene group.

  The hydrophilicity of the conjugate can be determined by comparing the hydrophilicity of the conjugate with the hydrophilicity of the unconjugated targeting agent (ie, the ligand or ligand unit), referred to as the hydrophilicity index . In certain embodiments, the retention time of the conjugate will not be delayed more than 2 minutes from the retention time of the unconjugated ligand, as determined as described in the Examples. In certain other embodiments, the retention time of the conjugate will not be delayed more than 1 minute from the retention time of the unconjugated ligand, as determined as described in the Examples. In certain other embodiments, the retention time of the conjugate will not be delayed more than 0.5 minutes from the retention time of the unconjugated ligand, as determined as described in the Examples. Referring to the examples, Example 3 discloses a preferred method for determining the hydrophilicity index of a conjugate. Instead, different hydrophobic interaction columns and / or methods are calibrated using the conjugate from Table 2 as a reference, and the reference conjugate mobility (elution of reference) on the selected column and / or method. Time) can be determined. The selected hydrophobic interaction column and / or the determined reference mobility on the method can then be used to calculate the hydrophilicity index of the test substance (as determined according to Example 3). To). For example, auristatin T-Glu-Dpr-MA, mc-MMAF and mc-vc-PABC-MMAE drug linker can be used to form a conjugate for use as a reference. In another example, auristatin T-Glu-Dpr-MA-h1F6 ADC, h1F6-mc-MMAF and h1F6-mc-vc-PABC-MMAE ADC can be used as a reference.

In view of the above, one group of embodiments provides a ligand-linker-drug conjugate comprising a ligand unit and a plurality of linker-drug units attached to the ligand unit. The linker unit includes, for example, a hydrophilic linker (L ^H ) assembly that includes a ligand binding component via a thioether linkage. The drug unit includes a cytotoxic agent having a binding component for connection to the linker unit. In another group of embodiments, a ligand-linker-drug conjugate is provided, wherein the linker moiety comprises a hydrophilic linker assembly and the drug unit comprises a cytotoxic agent.

  In a related embodiment, a method is provided for administering a ligand-linker-drug conjugate to a patient for treatment of a disease. The disease can be, for example, cancer or an autoimmune disease. The ligand-linker-drug conjugate is administered in a therapeutically effective amount and on a therapeutically effective schedule. In some embodiments, the conjugate dose is the same or less than the dose of a comparable two-loaded conjugate (administered on a comparable schedule). In some embodiments, the conjugate dose is the same or less than the dose of a comparable 4-loaded conjugate (administered on a comparable schedule). In some embodiments, the conjugate dose is the same or less than a comparable two-loaded conjugate dose, while the dosing schedule is the same or less frequent . In some embodiments, the conjugate dose is the same or less than a comparable 4-loaded conjugate dose, while the dosing schedule is the same or less frequent . In some further embodiments, the conjugate dose is lower and the dosing schedule is the same or less frequent than the dosing schedule of comparable two-loaded conjugates. In some further embodiments, the conjugate dose is lower and the dosing schedule is the same or less frequent than the dosing schedule of a comparable 4-loaded conjugate. The comparative conjugate can be, for example, the same ligand-drug-linker conjugate with 2 or 4 drug loadings.

In another group of embodiments, a drug-linker unit is provided, where the linker moiety has a hydrophilic linker (L ^H ) assembly having a ligand binding component (eg, a linked maleimide moiety) suitable for reacting with the ligand. including. In another group of embodiments, a ligand-linker conjugate is provided, wherein the linker moiety comprises a hydrophilic linker (L ^H ) assembly having features suitable for attachment and release of drug units.

In another group of embodiments, a method of making a ligand-linker-drug conjugate is provided. In some embodiments, the linker moiety comprises a hydrophilic linker (L ^H ) assembly having a linked maleimide moiety suitable for reacting with a ligand. In a further aspect, the linker moiety comprises a hydrophilic linker (L ^H ) assembly having features suitable for release of the drug unit (when attached).
Ligand-linker drug conjugate

In one aspect, the following formula:
A ligand-linker-drug conjugate having the formula: or a pharmaceutically acceptable salt or solvate thereof,
Where
L is a ligand that specifically binds to the target;
L ^A is a ligand binding component,
L ^H is a hydrophilic linker that is optionally branched, and each branch of L ^H has the formula -AA ₁ -R ^L1 -R ^L2 -R ^L3-
Have
Where
AA ₁ is a hydrophilic amino acid that forms a cleavable peptide bond with the C-terminus of the drug unit to which it is attached;
R ^L1 is optional, and when R ^L1 is absent exists vital R ^L2 and R ^L3, are selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
R ^L2 is optional, and when R ^L2 is not present there vital R ^L3, are selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
R ^L3 is optional, and when R ^L3 is present, is selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
The subscript p is an integer from 4 to about 20,
The subscript p ′ is an integer from 1 to 4,
D is the formula
Have
Where
Each of R ¹ and R ² is independently selected from the group consisting of hydrogen (H) and optionally substituted —C ₁ -C ₈ alkyl, provided that both R ³ and R ^{3 ′} are not H Except in cases, R ¹ and R ² are not both H,
R ³ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
R ^{3 ′} is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
At least one of R ³ and R ^{3 ′} is not H,
R ⁴ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
R ⁵ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
Or R ⁴ and R ⁵ together form a carbocyclic ring and the formula — (CR ^a R ^b ) _n — (wherein R ^a and R ^b are H and optionally substituted —C Independently selected from the group consisting of _{1 to} C ₈ alkyl, and n is selected from the group consisting of 2, 3, 4, 5 and 6.
R ⁶ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
R ⁷ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
Each R ⁸ is independently from the group consisting of H, —OH, optionally substituted —C ₁ -C ₈ alkyl, and optionally substituted —O— (C ₁ -C ₈ alkyl). Selected
R ¹² is H, optionally substituted —C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted —X ¹ aryl, optionally substituted— C ₃ ~C ₈ ^-X 1 in which carbocyclic ring, optionally substituted with - _(C 3 ~C ₈ carbocycle), _-C is optionally substituted 1 -C ₈ alkylene -NH _2, optionally Selected from substituted -C ₃ -C ₈ heterocycle and optionally substituted -X ^1- (C ₃ -C ₈ heterocycle);
Each X ¹ is independently —C ₁ -C ₁₀ alkylene,
The ligand-linker-drug conjugate has a hydrophilicity index that is equal to or less than 2;
Left and right lines L ^H, respectively, indicate a covalent bond to the drug unit and L ^A, wavy line each D represents a covalent bond to L ^H.

In a related aspect, the following formula
A ligand-linker-drug conjugate having the formula: or a pharmaceutically acceptable salt or solvate thereof,
Where
L is a ligand that specifically binds to the target;
L ^A is a ligand binding component,
L ^H is a hydrophilic linker that is optionally branched, and each branch of L ^H has the formula -AA ₁ -R ^L1 -R ^L2 -R ^L3-
Have
Where
AA ₁ is a hydrophilic amino acid that forms a cleavable peptide bond with the C-terminus of the drug unit to which it is attached;
When R ^L3 and the R ^L3 is absent, R ^L1 is selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
R ^L2 is optional, and when R ^L2 is not present there vital R ^L3, are selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
R ^L3 is optional, and when R ^L3 is present, is selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
The subscript p is an integer from 4 to about 20,
The subscript p ′ is an integer from 1 to 4,
D is the formula
Have
Where
Each of R ¹ and R ² is independently selected from the group consisting of hydrogen (H) and optionally substituted —C ₁ -C ₄ alkyl, provided that both R ³ and R ^{3 ′} are not H Except in cases, R ¹ and R ² are not both H,
R ³ is selected from the group consisting of H and optionally substituted —C ₁ -C ₄ alkyl;
R ^{3 ′} is selected from the group consisting of H and optionally substituted —C ₁ -C ₄ alkyl;
At least one of R ³ and R ^{3 ′} is not H,
R ⁴ is selected from the group consisting of H and optionally substituted —C ₁ -C ₄ alkyl;
R ⁵ is selected from the group consisting of H and optionally substituted —C ₁ -C ₄ alkyl;
Or R ⁴ and R ⁵ together form a carbocyclic ring and the formula — (CR ^a R ^b ) _n — (wherein R ^a and R ^b are H and optionally substituted —C Independently selected from the group consisting of _{1 to} C ₄ alkyl, and n is selected from the group consisting of 2, 3, 4, 5 and 6.
R ⁶ is selected from the group consisting of H and optionally substituted —C ₁ -C ₄ alkyl;
R ⁷ is selected from the group consisting of H and optionally substituted —C ₁ -C ₄ alkyl;
Each R ⁸ is independently from the group consisting of H, —OH, optionally substituted —C ₁ -C ₄ alkyl, and optionally substituted —O— (C ₁ -C ₄ alkyl). Selected
R ¹² is H, optionally substituted —C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted —X ¹ aryl, optionally substituted— C ₃ ~C ₈ ^-X 1 in which carbocyclic ring, optionally substituted with - _(C 3 ~C ₈ carbocycle), _-C is optionally substituted 1 -C ₈ alkylene -NH _2, optionally Selected from substituted -C ₃ -C ₈ heterocycle and optionally substituted -X ^1- (C ₃ -C ₈ heterocycle);
Each X ¹ is independently —C ₁ -C ₁₀ alkylene,
The ligand-linker-drug conjugate has a hydrophilicity index that is equal to or less than 2;
Left and right lines L ^H, respectively, indicate a covalent bond to the drug unit and L ^A, wavy line each D represents a covalent bond to L ^H.

  Various components of the ligand-linker-drug conjugates of Formulas I and I 'are provided in more detail below.

In some aspects, the ligand-linker-drug conjugate comprises a ligand unit and at least four linker-drug units, each of the ligand unit and the drug unit (s) being a hydrophilic linker (L ^H ). Joined by the linker unit (s) containing the assembly. In some further embodiments, the linker unit is attached to the ligand unit via a thioether bond. In some related embodiments, each linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) that is conjugated directly to the ligand unit via a thioether linkage.

In some aspects, the ligand-linker-drug conjugate comprises a ligand unit and at least 6 linker-drug units, each of the ligand unit and drug unit (s) being a hydrophilic linker (L ^H ). Joined by the linker unit (s) containing the assembly. In some further embodiments, the linker unit is attached to the ligand unit via a thioether bond. In some related embodiments, each linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) that is conjugated directly to the ligand unit via a thioether linkage.

In some aspects, the ligand-linker-drug conjugate comprises a ligand unit and at least 8 linker-drug units, each of the ligand unit and drug unit (s) being a hydrophilic linker (L ^H ). Joined by the linker unit (s) containing the assembly. In some further embodiments, the linker unit is attached to the ligand unit via a thioether bond. In some related embodiments, each linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) that is conjugated directly to the ligand unit via a thioether linkage.

In some aspects, the ligand-linker-drug conjugate comprises a ligand unit and at least 10 linker-drug units, each of the ligand unit and drug unit (s) being a hydrophilic linker (L ^H ). Joined by the linker unit (s) containing the assembly. In some further embodiments, the linker unit is attached to the ligand unit via a thioether bond. In some related embodiments, each linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) that is conjugated directly to the ligand unit via a thioether linkage.

In some aspects, the ligand-linker-drug conjugate comprises a ligand unit and at least 16 linker-drug units, each of the ligand unit and drug unit (s) being a hydrophilic linker (L ^H ). Joined by the linker unit (s) containing the assembly. In some further embodiments, the linker unit is attached to the ligand unit via a thioether bond. In some related embodiments, each linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) that is conjugated directly to the ligand unit via a thioether linkage.
Drug unit

Referring to the drug units of formula I and I ′, in some embodiments, R ¹² is H, optionally substituted —C ₁ -C ₈ alkyl, optionally substituted aryl, optional Optionally substituted —X ¹ aryl, optionally substituted —C _{3 to} C ₈ carbocycle, optionally substituted —X ¹ — (C _{3 to} C ₈ carbocycle), optional —C ₁ -C ₈ alkylene-NH ₂ substituted with, optionally substituted —C ₃ -C ₈ heterocycle and optionally substituted —X ^1- (C ₃ -C ₈ complex) Ring).

In some related embodiments, R ¹² is not a side chain of phenylalanine or a side chain of proline. In some further related embodiments, R ¹² is not a phenylalanine side chain, a methionine side chain, a tryptophan side chain, or a proline side chain.

In some embodiments, R ¹² is selected from the side chains of natural L-amino acids other than proline and glycine. In some further embodiments, R ¹² is selected from the side chains of natural L-amino acids other than proline, glycine and phenylalanine. In some further embodiments, R ¹² is selected from the side chains of natural L-amino acids other than proline, glycine, tryptophan, methionine and phenylalanine.

In some further embodiments, R ¹² is threonine, serine, asparagine, aspartic acid, glutamine, glutamic acid, homoserine, hydroxyvaline, furylalanine, threonine (PO ₃ H ₂ ), pyrazolylalanine, triazolylalanine and thiayl. Selected from the side chain of the group of hydrophilic amino acids consisting of zolylalanine.

In some embodiments, R ¹² is the threonine side chain.

An exemplary drug unit has the formula: or a pharmaceutically acceptable salt thereof, and the wavy line indicates the site of attachment to the linker unit. In some exemplary units, the drug unit is dimethyl- or monomethyl-auristatin F as shown below:
Or a pharmaceutically acceptable salt or solvate thereof.

Other exemplary drug units are the dimethyl or monomethyl form of auristatin T
Or a pharmaceutically acceptable salt or solvate thereof.
Linker unit

Referring to the linker unit of the formula I and I ', in some embodiments, L ^A is covalently linked to the sulfur atom of the ligand. In some embodiments, the sulfur atom is a sulfur atom of a cysteine residue that can form an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is the sulfur atom of a cysteine residue introduced into the ligand unit (eg, by site-directed mutagenesis or a chemical reaction). In a further embodiment, a sulfur atom L ^A is attached is cysteine residues forming the interchain disulfide bonds of the antibody, and were introduced into the Ligand unit (e.g., by site-directed mutagenesis or chemical reaction) cysteine Selected from residues.

AA ₁ forms a cleavable bond with the drug unit. In embodiments where AA ₁ is bound to an amino acid of the drug unit, AA ₁ forms a cleavable peptide bond with the drug unit. A cleavable peptide bond is susceptible to cleavage by a protease when the conjugate reaches its target site. In some embodiments, AA ₁ is selected from the group consisting of hydrophilic amino acids, typically glycine, and the L-form of aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine. It is an amino acid. In some embodiments, AA ₁ is glutamate.

In embodiments where R ^L1 is present and is a hydrophilic amino acid, it is glycine; aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine in L or D form; —NH—CH ( R ^a ) —CO—; and —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH _{2. CH 2 CH 2 NH 2, -CH} 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, and _{-CH 2 CH 2 CH 2 CH 2} CO 2 H It is selected from, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 _{H -, - CH 2 CH 2} C (O) -, - CH 2 CH 2 CH 2 C (O) -, and _{-CH 2 CH 2 CH 2 CH 2} C (O) - is selected from. In some further embodiments, R ^L1 is D-amino acid of aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; glycine; —NH—CH (R ^a ) —C (O) — And selected from the group consisting of —NH—CH (COOH) —R ^b —, wherein R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ OH, —CH ₂ CH ₂ CH ₂ CO ₂ H, and —CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H, wherein R ^b is — _{CH 2 NH -, - CH 2} CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 C (O _{-, - CH 2 CH 2 CH} 2 C (O) -, and _{-CH 2 CH 2 CH 2 CH 2} C (O) - is selected from.

In embodiments where R ^L1 is present and is optionally substituted alkylene, it is —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl), —NH It may be C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents selected from ₂ or —NH (C ₁ -C ₃ alkyl). In some embodiments, R ^L1 is ethylenediamine, —NH—CH (COOH) —CH ₂ —NH—, or —C (O) —CH (CH ₂ NH ₂ ) —.

In embodiments where R ^L2 is present and is a hydrophilic amino acid, it is glycine; aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine in the L or D form; —NH—CH ( R ^a ) —C (O) —; and —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , _{-CH 2 CH 2 CH 2 NH 2} , -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, and _-CH 2 _CH 2 _CH 2 CH ₂ is selected from CO ₂ H, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH ₂ NH—, —CH ₂ CH ₂ C (O) —, —CH ₂ CH ₂ CH ₂ C (O) —, and —CH ₂ CH ₂ CH ₂ CH ₂ C (O) —. In some further embodiments, when R ^L2 is present, it is an aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine D amino acid; glycine; —NH—CH (R ^a ) — C (O) —; and —NH—CH (COOH) —R ^b —, wherein R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH _{2. NH 2, -CH 2 CH 2 CH} 2 CH 2 NH 2, is selected from _{-CH 2 CH 2 OH, -CH 2} CH 2 CH 2 CO 2 H, and _{-CH 2 CH 2 CH 2 CH 2} CO 2 H, R ^b _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - _{H 2 CH 2 C (O)} -, - CH 2 CH 2 CH 2 C (O) -, and _{-CH 2 CH 2 CH 2 CH 2} C (O) - is selected from.

In embodiments wherein R ^L2 is present and is optionally substituted alkylene, it is —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl) —, — It may be C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents selected from NH ₂ or —NH (C ₁ -C ₃ alkyl). In some embodiments, R ^L2 is ethylenediamine, —NH—CH (COOH) —CH ₂ —NH—, or —C (O) —CH (CH ₂ NH ₂ ) —.

In embodiments where R ^L3 is present and is a hydrophilic amino acid, it is glycine; aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine, L-form or D-form; R ^a ) —C (O) —; and —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , _{-CH 2 CH 2 CH 2 NH 2} , -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, and _-CH 2 _CH 2 _CH 2 CH ₂ is selected from CO ₂ H, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH ₂ NH—, —CH ₂ CH ₂ C (O) —, —CH ₂ CH ₂ CH ₂ C (O) —, and —CH ₂ CH ₂ CH ₂ CH ₂ C (O) —.

In some further embodiments, when R ^L3 is present, it is a D amino acid of aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; glycine; —NH—CH (R ^a ) — C (O) —; and —NH—CH (COOH) —R ^b —, wherein R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH _{2. NH 2, -CH 2 CH 2 CH} 2 CH 2 NH 2, is selected from _{-CH 2 CH 2 OH, -CH 2} CH 2 CH 2 CO 2 H, and _{-CH 2 CH 2 CH 2 CH 2} CO 2 H, R ^b _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - _{H 2 CH 2 C (O)} -, - CH 2 CH 2 CH 2 C (O) -, and _{-CH 2 CH 2 CH 2 CH 2} C (O) - is selected from.

In embodiments where R ^L3 is present and is optionally substituted alkylene, it is —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl) —, —NH. It may be C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents selected from ₂ or —NH (C ₁ -C ₃ alkyl). In some embodiments, R ^L1 is ethylenediamine, —NH—CH (COOH) —CH ₂ —NH—, or —C (O) —CH (CH ₂ NH ₂ ) —.

In some embodiments above, AA ₁ is present and R ^L1 , R ^L2 and R ^L3 are absent.

In some embodiments above, AA ₁ is present, R ^L1 is present, and R ^L2 and R ^L3 are absent.

In some embodiments above, AA ₁ is present, R ^L1 is present, R ^L2 is present, and R ^L3 is absent.

In some embodiments above, AA ₁ is present, R ^L1 is present, R ^L2 is present, and R ^L3 is present.

In some embodiments above, AA ₁ is a hydrophilic amino acid, at least one of R ^L1 , R ^L2 and R ^L3 is present and optionally substituted as described above. Alkylene.

In some embodiments above, AA ₁ is glutamate, at least one of R ^L1 , R ^L2, and R ^L3 is present and is optionally substituted alkylene as described above It is.

In some embodiments above, AA ₁ is glutamate, R ^L1 is a hydrophilic amino acid, at least one of R ^L2 and R ^L3 is present, and any as described above Alkylene is optionally substituted.

In some embodiments above, AA ₁ and R ^L1 are hydrophilic amino acids, at least one of R ^L2 and R ^L3 is present and optionally substituted as described above. Alkylene.

In some embodiments above, AA ₁ is a hydrophilic amino acid and R ^L1 and optionally R ^L2 are optionally substituted alkylene as described above.

In some embodiments of the, ^{L H} is glycine dipeptide (Gly-Gly) is also a tripeptide is also not included tetrapeptide. In some embodiments, L ^H does not include the peptide Asn- (D) Lys.

In some embodiments, L ^H comprises a modified peptide having 2-4 amino acids. The modified peptide has an amino acid (AA ₁ ) selected at position ₁ to optimize the release of the drug unit (eg, by protease cleavage via an amide peptide bond). In one or both positions, R ^L1 and R ^L2 reverse the orientation of the typical N to C linkage of the peptide (form an amide bond) and the last amino acid (eg, R ^L2 or R ^L3 ), Which contains an α-amino group protected as a maleimide prior to attachment of the ligand unit. An amino acid having an inverted N to C linkage is linked to the next group through its side chain. In some embodiments, the amino acid is an alpha amino acid. In other embodiments, it can be a beta or gamma amino acid. In some of these embodiments, the side chain is —CH ₂ NH ₂ —, —CH ₂ CH ₂ NH ₂ —, —CH ₂ CH ₂ CH ₂ NH ₂ —, and —CH ₂ CH ₂ CH ₂ CH _2. NH ₂ - is selected from.

In some embodiments of L ^H , according to any of the above embodiments, the amino acid having an inverted N to C linkage (R ^L1 ) is bound to R ^L2 or R ^L3 , and R ^L2 Or R ^L3 is a hydrophilic amino acid or an optionally substituted alkylene.

In some embodiments of L ^H, by any of the above embodiments, the amino acid having a connection to C from reversed the N (R ^L1) is attached to R ^L2, R ^L2 is optionally Alkylene which is optionally substituted.

In some further embodiments, L ^H is a hydrophilic cleavable linker and each branch is of the formula
In which R ²¹ is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, —CH ₂ CH ₂ OH, —CH ₂ CO ₂ H, —CH ₂ CH ₂ CO Selected from ₂ H, —CH ₂ CH ₂ CH ₂ CO ₂ H and —CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H, R ²² is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH Selected from ₂ OH, and —CH ₂ CH ₂ OH. The left and right wavy lines indicate the drug unit and the binding to the L ^A or L ^H branch, respectively.

In a further embodiment, L ^H or a branch thereof is of the formula
Wherein R ²² is selected from —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, and —CH ₂ CH ₂ OH. In some embodiments, R ²² is selected from —CH ₂ NH ₂ and —CH ₂ CH ₂ NH ₂ . The left and right wavy lines indicate the drug unit and the binding to the L ^A or L ^H branch, respectively.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate binding to the Drug unit and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have The left and right wavy lines indicate the drug unit and the binding to the L ^A or L ^H branch, respectively.

In certain embodiments, L ^H or a branch thereof is of the formula
Have The left and right wavy lines indicate the drug unit and the binding to the L ^A or L ^H branch, respectively.

In certain embodiments, L ^H or a branch thereof is of the formula
Have The left and right wavy lines indicate the drug unit and the binding to the L ^A or L ^H branch, respectively.

In certain embodiments, L ^H or a branch thereof is of the formula
Have The left and right wavy lines indicate the drug unit and the binding to the L ^A or L ^H branch, respectively.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate binding to the Drug unit and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have The left and right wavy lines indicate the drug unit and the binding to the L ^A or L ^H branch, respectively.

In some further embodiments above, L ^H is of the formula
A branched hydrophilic linker having
In the formula, each R ³¹ is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, —CH ₂ CH ₂ OH, —CH ₂ CO ₂ H, —CH ₂ CH ₂ CO ₂ H, _{-CH 2 CH 2 CH 2 CO 2} H, and is selected from the group consisting of _{-CH 2 CH 2 CH 2 CH 2} CO 2 H ^{independently} each bar adjacent to the ^{R 31,} binding to _{D E} units The vertical dashed line indicates the binding to the ligand unit.

In some further embodiments above, L ^H is of the formula
A branched hydrophilic linker having
In the formula, each bar represents binding to a drug unit and a vertical dashed line represents binding to a ligand unit.

The L ^A subunit, described in more detail below.

Referring back to the ligand-linker-drug conjugates of formulas I and I ′, in some further embodiments, the ligand-linker-drug conjugate is
Having an expression selected from
In the formula, S refers to the sulfur atom of the ligand.

Certain embodiments are described below.
(Wherein S refers to the sulfur atom of the ligand), or a pharmaceutically acceptable salt or solvate thereof.

In yet another aspect, the following formula:
A ligand-linker-drug conjugate having
Where
L is a ligand that specifically binds to the target;
L ^A is a ligand binding component,
L ^H is an optional hydrophilic linker, and each branch of L ^H has the formula -AA ₁ -R ^L1 -R ^L2 -R ^L3-
Have
Where
AA ₁ is a hydrophilic amino acid that forms a cleavable bond with _DE ,
R ^L1 is optional, and when R ^L1 is absent exists vital R ^L2 and R ^L3, are selected from alkylene substituted with hydrophilic amino acids, or optionally may share L ^A and atoms,
R ^L2 is optional, and when R ^L2 is not present there vital R ^L3, are selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
R ^L3 is optional, and when R ^L3 is present, is selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
L ^A is a ligand binding component,
The subscript p is an integer from 4 to about 20,
The subscript p ′ is an integer from 1 to 4,
Ligand - linker - drug conjugate has a hydrophilic index is less than or 2 equal to 2, the left and right of the line of L ^H, respectively, indicate a covalent bond to the D _E units and L ^A,
_DE is an effector moiety as further described herein.

  Various components of the ligand-linker-drug conjugate of Formula II are provided in more detail below.

In some embodiments, the ligand - linker - drug conjugate comprises a Ligand unit and at least four linker -D _E units, each of the Ligand unit and D _E unit (s), hydrophilic linker (L ^H ) Joined by a linker unit containing assembly. In some further embodiments, the linker unit is attached to the ligand unit via a thioether bond. In some related embodiments, the linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) that is conjugated directly to the ligand unit via a thioether linkage.

In some embodiments, the ligand - linker - drug conjugate comprises a Ligand unit and at least six Linker -D _E units, each of the Ligand unit and D _E unit (s), hydrophilic linker (L ^H ) Joined by a linker unit containing assembly. In some further embodiments, the linker unit is attached to the ligand unit via a thioether bond. In some related embodiments, the linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) that is conjugated directly to the ligand unit via a thioether linkage.

In some embodiments, the ligand - linker - drug conjugate comprises a Ligand unit and at least eight linker -D _E units, each of the Ligand unit and D _E unit (s), hydrophilic linker (L ^H ) Joined by a linker unit containing assembly. In some further embodiments, the linker unit is attached to the ligand unit via a thioether bond. In some related embodiments, the linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) that is conjugated directly to the ligand unit via a thioether linkage.

In some aspects, the ligand-linker-drug conjugate comprises a ligand unit and at least 10 linker-D _E units, each of the ligand unit and D _E unit (s) being a hydrophilic linker (L ^H ) Joined by a linker unit containing assembly. In some further embodiments, the linker unit is attached to the ligand unit via a thioether bond. In some related embodiments, the linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) that is conjugated directly to the ligand unit via a thioether linkage.

In some aspects, the ligand-linker-drug conjugate comprises a ligand unit and at least 16 linker-D _E units, each of the ligand unit and D _E unit (s) being a hydrophilic linker (L ^H ) Joined by a linker unit containing assembly. In some further embodiments, the linker unit is attached to the ligand unit via a thioether bond. In some related embodiments, the linker unit further comprises a hydrolyzed succinimide ring (or succinic acid) that is conjugated directly to the ligand unit via a thioether linkage.

Referring to the Linker unit of Formula II, in some embodiments, L ^A is covalently linked to the sulfur atom of the ligand. In some embodiments, the sulfur atom is a sulfur atom of a cysteine residue that forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is the sulfur atom of a cysteine residue introduced into the ligand unit (eg, by site-directed mutagenesis or a chemical reaction). In a further embodiment, a sulfur atom L ^A is attached (s), cysteine residues forming inter-chain disulfide bonds of antibodies, and is introduced into the Ligand unit (e.g., site-directed mutagenesis or chemically Selected from cysteine residues (depending on the reaction).

AA ₁ forms a cleavable bond with the _DE unit. In embodiments where AA ₁ is bound to an amino acid of _{D E} unit, AA ₁ forms a cleavable peptide bond with the _{D E} units. A cleavable peptide bond is susceptible to cleavage by a protease when the conjugate reaches its target site. In other embodiments, AA ₁ forms an amide bond with the binding site of the effector moiety (D _E ) that is susceptible to cleavage (eg, by a protease) when the conjugate reaches its targeted site. In some embodiments of Formula II, AA ₁ is from the group consisting of a hydrophilic amino acid, typically glycine, and the L form of aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine. The natural amino acid selected. In some embodiments, AA ₁ is glutamate.

In embodiments where R ^L1 is present and is a hydrophilic amino acid, it is glycine; aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine in L or D form; —NH—CH ( R ^a ) —C (O) —; and —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , _{-CH 2 CH 2 CH 2 NH 2} , -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, and _-CH 2 _CH 2 _CH 2 CH ₂ is selected from CO ₂ H, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH ₂ NH—, —CH ₂ CH ₂ C (O) —, —CH ₂ CH ₂ CH ₂ C (O) —, and —CH ₂ CH ₂ CH ₂ CH ₂ C (O) —. In some further embodiments, R ^L1 is D-amino acid of aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; glycine; —NH—CH (R ^a ) —C (O) — And selected from the group consisting of —NH—CH (COOH) —R ^b —, wherein R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ OH, —CH ₂ CH ₂ CH ₂ CO ₂ H, and —CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H, wherein R ^b is — _{CH 2 NH -, - CH 2} CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 C (O _{-, - CH 2 CH 2 CH} 2 C (O) -, and _{-CH 2 CH 2 CH 2 CH 2} C (O) - is selected from.

In embodiments wherein R ^L1 is present and is optionally substituted alkylene, it is —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl) —, — It may be C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents selected from NH ₂ or —NH (C ₁ -C ₃ alkyl). In some embodiments, R ^L1 is ethylenediamine, —NH—CH (COOH) —CH ₂ —NH—, or —C (O) —CH (CH ₂ NH ₂ ) —.

In embodiments where R ^L2 is present and is a hydrophilic amino acid, it is glycine; aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine in the L or D form; —NH—CH ( R ^a ) —C (O) —; and —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , _{-CH 2 CH 2 CH 2 NH 2} , -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, and _-CH 2 _CH 2 _CH 2 CH ₂ is selected from CO ₂ H, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH ₂ NH—, —CH ₂ CH ₂ C (O) —, —CH ₂ CH ₂ CH ₂ C (O) —, and —CH ₂ CH ₂ CH ₂ CH ₂ C (O) —.

In some further embodiments, when R ^L2 is present, it is an aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine D amino acid; glycine; —NH—CH (R ^a ) — C (O) —; and —NH—CH (COOH) —R ^b —, wherein R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH _{2. NH 2, -CH 2 CH 2 CH} 2 CH 2 NH 2, is selected from _{-CH 2 CH 2 OH, -CH 2} CH 2 CH 2 CO 2 H, and _{-CH 2 CH 2 CH 2 CH 2} CO 2 H, R ^b _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - _{H 2 CH 2 C (O)} -, - CH 2 CH 2 CH 2 C (O) -, and _{-CH 2 CH 2 CH 2 CH 2} C (O) - is selected from.

In embodiments wherein R ^L2 is present and is optionally substituted alkylene, it is —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl) —, — It may be C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents selected from NH ₂ or —NH (C ₁ -C ₃ alkyl). In some embodiments, R ^L2 is ethylenediamine, —NH—CH (COOH) —CH ₂ —NH—, or —C (O) —CH (CH ₂ NH ₂ ) —.

In embodiments where R ^L3 is present and is a hydrophilic amino acid, it is glycine; aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine, L-form or D-form; R ^a ) —C (O) —; and —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , _{-CH 2 CH 2 CH 2 NH 2} , -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, and _-CH 2 _CH 2 _CH 2 CH ₂ is selected from CO ₂ H, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH ₂ NH—, —CH ₂ CH ₂ C (O) —, —CH ₂ CH ₂ CH ₂ C (O) —, and —CH ₂ CH ₂ CH ₂ CH ₂ C (O) —.

In some further embodiments, when R ^L3 is present, it is a D amino acid of aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; glycine; —NH—CH (R ^a ) — As well as —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH _{2. NH 2, -CH 2 CH 2 CH} 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, H 2 CO 2 H and _{-CH 2 CH 2 CH 2 CH 2} CO 2 It is selected from H, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH ₂ NH—, —CH ₂ CH ₂ C (O) —, —CH ₂ CH ₂ CH ₂ C (O) —, and —CH ₂ CH ₂ CH ₂ CH ₂ C (O) —.

In embodiments where R ^L3 is present and is optionally substituted alkylene, it is —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl) —, —NH. It may be C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents selected from ₂ or —NH (C ₁ -C ₃ alkyl). In some embodiments, R ^L3 is ethylenediamine, —NH—CH (COOH) —CH ₂ —NH—, or —C (O) —CH (CH ₂ NH ₂ ) —.

In some embodiments above, AA ₁ is present and R ^L1 , R ^L2 and R ^L3 are absent.

In some embodiments above, AA ₁ is present, R ^L1 is present, and R ^L2 and R ^L3 are absent.

In some embodiments above, AA ₁ is present, R ^L1 is present, R ^L2 is present, and R ^L3 is absent.

In some embodiments above, AA ₁ is present, R ^L1 is present, R ^L2 is present, and R ^L3 is present.

In some further embodiments above, AA ₁ is a hydrophilic amino acid, at least one of R ^L1 , R ^L2 and R ^L3 is present and optionally substituted as described above. Alkylene.

In some embodiments above, AA ₁ is glutamate, at least one of R ^L1 , R ^L2, and R ^L3 is present and is optionally substituted alkylene as described above It is.

In some embodiments above, AA ₁ is glutamate, R ^L1 is a hydrophilic amino acid, at least one of R ^L2 and R ^L3 is present, and any as described above Alkylene is optionally substituted.

In some further embodiments above, AA ₁ and R ^L1 are hydrophilic amino acids, at least one of R ^L2 and R ^L3 is present and optionally substituted as described above. Alkylene.

In some embodiments above, AA ₁ is a hydrophilic amino acid and R ^L1 and optionally R ^L2 are optionally substituted alkylene as described above.

In some embodiments of the, ^{L H} is glycine dipeptide (Gly-Gly) is also a tripeptide is also not included tetrapeptide. In some embodiments, L ^H does not include the peptide Asn- (D) Lys.

In some embodiments, L ^H comprises a modified peptide having 2-4 amino acids. The modified peptide has, at position 1, an amino acid (AA ₁ ) that is selected to optimize the release of the _DE unit (eg, by protease cleavage via an amide peptide bond). In one or both positions, R ^L1 and R ^L2 reverse the typical N to C linkage orientation of the peptide and promote the binding of the last amino acid (eg, R ^L2 or R ^L3 ). Which contains an α-amino group protected as a maleimide prior to attachment of the ligand unit. An amino acid having an inverted N to C linkage is linked to the next group through its side chain. In some embodiments, the amino acid is an alpha amino acid. In other embodiments, it can be a beta or gamma amino acid. In some embodiments, the side _{chains, -CH 2 NH 2, -CH 2} CH 2 NH 2, -CH 2 CH 2 CH 2 NH 2 - Selection, and _{-CH 2 CH 2 CH 2 CH 2} NH 2 Is done.

In some embodiments of L ^H , according to any of the above embodiments, the amino acid having an inverted N to C linkage (R ^L1 ) is bound to R ^L2 or R ^L3 , and R ^L2 Or R ^L3 is a hydrophilic amino acid or an optionally substituted alkylene.

In some embodiments of L ^H, by any of the above embodiments, the amino acid having a connection to C from reversed the N (R ^L1) is attached to R ^L2, R ^L2 is optionally Alkylene which is optionally substituted.

In some further embodiments, L ^H is a hydrophilic cleavable linker and each branch is of the formula
In which R ²¹ is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, —CH ₂ CH ₂ OH, —CH ₂ CO ₂ H, —CH ₂ CH ₂ CO ₂ H, —CH ₂ CH ₂ CH ₂ CO ₂ H, and —CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H, R ²² is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , — CH ₂ OH, is selected from _-CH 2 _CH 2 OH. The left and right squiggly lines indicate the D _E unit and the connection to the L ^A or L ^H branch, respectively.

In a further embodiment, L ^H or a branch thereof is of the formula
Wherein R ²² is selected from —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, and —CH ₂ CH ₂ OH. In some embodiments, R ² is selected from —CH ₂ NH ₂ and —CH ₂ CH ₂ NH ₂ . Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have The left and right squiggly lines indicate the D _E unit and the connection to the L ^A or L ^H branch, respectively.

In certain embodiments, L ^H or a branch thereof is of the formula
Have The left and right squiggly lines indicate the D _E unit and the connection to the L ^A or L ^H branch, respectively.

In certain embodiments, L ^H or a branch thereof is of the formula
Have The left and right squiggly lines indicate the D _E unit and the connection to the L ^A or L ^H branch, respectively.

In certain embodiments, L ^H or a branch thereof is of the formula
Have The left and right squiggly lines indicate the D _E unit and the connection to the L ^A or L ^H branch, respectively.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have The left and right squiggly lines indicate the D _E unit and the connection to the L ^A or L ^H branch, respectively.

In certain embodiments, L ^H or a branch thereof is of the formula
Have The left and right squiggly lines indicate the D _E unit and the connection to the L ^A or L ^H branch, respectively.

In some further embodiments above, L ^H is of the formula
A branched hydrophilic linker having
In the formula, each R ³¹ is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, —CH ₂ CH ₂ OH, —CH ₂ CO ₂ H, —CH ₂ CH ₂ CO ₂ H, _{-CH 2 CH 2 CH 2 CO 2} H, and is selected from the group consisting of _{-CH 2 CH 2 CH 2 CH 2} CO 2 H ^{independently} each bar adjacent to the ^{R 31,} binding to _{D E} units The vertical dashed line indicates the binding to the ligand unit.

In some further embodiments above, L ^H is of the formula
A branched hydrophilic linker having
In the formula, each of the bars indicates binding to the _DE unit, and a vertical dashed line indicates binding to the ligand unit.

In some further embodiments described above, the branched hydrophilic linker is of the formula
Have

Referring back to the ligand-linker-drug conjugate of Formula II, in some further embodiments, the ligand-linker-drug conjugate is
Having an expression selected from
In the formula, S refers to the sulfur atom of the ligand.
L ^A -ligand binding component

Formula I, again referring to I 'and II (above), ^{L A} is a ligand binding component. In some embodiments, L ^A may be a maleimide or hydrolyzed maleimide or succinimide group (hereinafter exemplified as succinic acid moiety). In some embodiments L _A is attached to the Ligand unit, which is a hydrolyzed maleimide or succinimide group (exemplified as succinic acid moiety). Thus, in some embodiments, L ^A is formula
Where the wavy line indicates the point of attachment to L ^H and \\ indicates the point of attachment to L, the ligand unit.

In other embodiments, L ^A is of the formula
Where the wavy line indicates the point of attachment to L ^H and \\ indicates the point of attachment to L, the ligand unit.

In the context of the present invention, in some embodiments (as shown above), L ^A is the evidence of maleimide groups are used in the binding of the ligand moiety. Design of L ^H and L ^A is to enable easy addition of Ligand unit, to provide additional carboxylic acid groups, whereby the ligand - increasing the hydrophilicity of the drug conjugate. Still further, maleimide nitrogen becomes the α-amine of amino acid 3 (with respect to L ^H ).
L-ligand

  Referring back to Formulas I, I 'and II, the ligand unit (L-) is a targeting agent that specifically binds to the targeting moiety. The ligand can specifically bind to a cellular component or other target molecule of interest. The target moiety or target is typically on the cell surface. In some embodiments, the ligand unit acts to deliver the drug unit to the specific target cell population with which the ligand unit interacts. Ligand includes, but is not limited to, proteins, polypeptides and peptides, and non-proteinaceous agents such as carbohydrates. Suitable ligand units include, for example, antibodies, such as full-length (intact) antibodies, and antigen-binding fragments thereof.

  In embodiments where the ligand unit is a non-antibody targeting agent, it can be a peptide or polypeptide, or a non-proteinaceous molecule. Examples of such targeting agents include interferons, lymphokines, hormones, growth factors and colony stimulating factors, vitamins, nutrient-transport molecules (including but not limited to transferrin), or any other cell binding molecule or Contains substances.

In some embodiments, L ^A is covalently linked to the sulfur atom of the ligand. In some embodiments, the sulfur atom is a sulfur atom of a cysteine residue that forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is the sulfur atom of a cysteine residue introduced into the ligand unit (eg, by site-directed mutagenesis or a chemical reaction). In a further embodiment, a sulfur atom L ^A is attached is cysteine residues forming the interchain disulfide bonds of the antibody, and were introduced into the Ligand unit (e.g., by site-directed mutagenesis or chemical reaction) cysteine Selected from residues. In some embodiments, cysteine residues are EU-indexed as in Kabat (Kabat EA et al. (1991) Sequences of Proteins of Immunological Intersect, 5th edition, NIH Publication 91, 3242). It is introduced into the Fc region at position 239 according to the numbering system.

In some embodiments, the Ligand unit forms a bond with a maleimide present on L ^H via the sulfhydryl group of the ligand, to form the thio-substituted succinimide. A sulfhydryl group can be present on a ligand in its native state, eg, a naturally occurring antibody, or can be introduced into a ligand by chemical modification. Hydrolysis of the remaining succinimide produces L ^A portion.

  In one aspect, the ligand unit has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. Reagents that can be used to modify lysine include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Trout reagent).

  In another embodiment, the ligand unit can have one or more carbohydrate groups that can be chemically modified to have one or more sulfhydryl groups.

  In another embodiment, sulfhydryl groups can be generated by reduction of interchain disulfides. Thus, in some embodiments, the linker unit is conjugated to a cysteine residue of the reduced interchain disulfide.

  In another embodiment, the sulfhydryl group is chemically introduced into the antibody, for example, by introduction of a cysteine residue. Thus, in some embodiments, the linker unit is conjugated to an introduced cysteine residue.

  Useful non-immunoreactive protein ligands, polypeptide ligands, or peptide ligands include, but are not limited to, non-immunoreactive protein ligands, polypeptide ligands, or peptide ligands instead of antibodies. Transferrin, epidermal growth factor (“EGF”), bombesin, gastrin, gastrin releasing peptide, platelet derived growth factor, IL-2, IL-6, transforming growth factor (“TGF”), eg, TGF-α and TGF -Β, vaccinia growth factor (“VGF”), insulin and insulin-like growth factors I and II, somatostatin, lectins, and apoproteins from low density lipoproteins.

  A particularly preferred ligand is an antibody. In fact, in any of the embodiments described herein, the ligand unit may be an antibody. Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Useful monoclonal antibodies are homogeneous populations of antibodies against specific antigenic determinants (eg, against cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). Monoclonal antibodies (mAbs) against the antigen of interest can be prepared by using any technique known in the art that achieves production of antibody molecules by continuous cell lines in culture.

  Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, or chimeric human-mouse (or other species) monoclonal antibodies. Antibodies include full length antibodies and antigen binding fragments thereof. Human monoclonal antibodies can be made by any of a number of techniques known in the art (eg, Teng et al., 1983, Proc. Natl. Acad. Sci. USA., 80: 7308-7312; Kozbor). 1983, Immunology Today, 4: 72-79; and Olsson et al., 1982, Meth. Enzymol., 92: 3-16).

  An antibody is an antigen-binding fragment, derivative or analog of an antibody that immunospecifically binds to a target cell (eg, a cancer cell antigen, viral antigen, or microbial antigen), or other antibody that binds to a tumor cell or matrix. possible. In this regard, “antigen binding” means that a fragment, derivative or analog can specifically bind to a target moiety. Specifically, in an exemplary embodiment, the idiotypic antigenicity of an immunoglobulin molecule is enhanced by deletion of a framework and CDR sequences that are C-terminal to a CDR sequence that specifically recognizes the antigen. be able to. In order to determine which CDR sequences bind to an antigen, synthetic peptides containing CDR sequences can be synthesized in a binding assay involving an antigen by any binding assay method known in the art (eg, the BIA core assay). (Eg, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, National Institute of Health, Bethesda, Md; Kabat E et al., 1980, J. Immun). : See pages 961-969).

Other useful antibodies include, but are not limited to, F (ab ′) ₂ fragment, Fab fragment, Fvs, single chain antibody, bispecific antibody, trispecific antibody, tetraspecific antibody, scFv, scFv-FV Or an antigen-binding fragment of an antibody, such as any other molecule derived from and having the same specificity as an antibody.

  In addition, recombinant antibodies that contain both human and non-human portions, eg, chimeric antibodies and humanized monoclonal antibodies, that can be made using standard recombinant DNA techniques are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal and a human immunoglobulin constant region. (See, eg, US Pat. No. 4,816,567, which is incorporated herein by reference in its entirety; and US Pat. No. 4,816,397.) Humanized antibodies are non-human. An antibody molecule from a non-human species having one or more complementarity determining regions (CDRs) from the species and a framework region derived from a human immunoglobulin molecule. (See, eg, US Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies are known in the art. By recombinant DNA technology, for example, WO 87/02671; European Patent Application Publication No. 0184187; European Patent Application Publication No. 0171496, each of which is incorporated herein by reference in its entirety. European Patent Application Publication No. 0173494; International Publication No. WO86 / 01533; US Patent No. 4,816,567; European Patent Application Publication No. 012023; Berter et al., 1988, Science, 240: 1041-1043. Liu et al., 1987, Proc. Natl. Acad. Sci. USA, 84: 3439-3443; Liu et al., 1987, J. MoI. Immunol. 139: 3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA, 84: 214-218; Nishimura et al., 1987, Cancer. Res. 47: 999-1005; Wood et al., 1985, Nature, 314: 446-449; and Shaw et al., 1988, J. MoI. Natl. Cancer Inst. Morrison, 1985, Science, 229: 1202-1207; Oi et al., 1986, BioTechniques, 4: 214; US Pat. No. 5,225,539; Jones et al. 1986, Nature 321: 552-525; Verhoeyan et al. 1988 Science 239: 1534; and Beidler et al. Immunol. 141: 4053-4060.

  Using transgenic mice, where fully human antibodies are particularly desirable and cannot express endogenous immunoglobulin heavy and light chain variable region genes, but can express human heavy and light chain variable region genes Can be produced.

  The antibody can have modifications (eg, substitutions, deletions and / or additions) at amino acid residues that interact with the Fc receptor. In particular, the antibody may have modifications at amino acid residues that have been identified as being involved in the interaction between the anti-Fc domain and the FcRn receptor (eg, in its entirety herein by reference). See incorporated International Publication No. WO 97/34631). The antibody can also have modifications in amino acid residues that have been identified as being involved in the interaction between the anti-Fc domain and Fc gamma receptor III.

  Antibodies that are immunospecific for a cancer cell antigen can be obtained commercially or can be produced by any method known to those skilled in the art, such as chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for a cancer cell antigen can be obtained, for example, from the GenBank database or similar databases, literature sources, or by routine cloning and sequencing.

  In certain embodiments, antibodies for the treatment of cancer can be used. Antibodies immunospecific for a cancer cell antigen can be obtained commercially or can be produced by any method known to those of skill in the art, such as recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for a cancer cell antigen can be obtained, for example, from the GenBank database or similar databases, literature sources, or by routine cloning and sequencing.

  In another specific embodiment, antibodies for the treatment of autoimmune diseases are used according to the compositions and methods of the invention. Antibodies that are immunospecific for antigens of cells involved in the production of autoimmune antibodies can be obtained from any institution (eg, a university scientist or company) or known to those skilled in the art It can be produced by any method, such as chemical synthesis or recombinant expression techniques.

  In certain embodiments, useful antibodies can bind to a receptor or receptor complex. A receptor or receptor complex includes, for example, an immunoglobulin gene superfamily member, a TNF receptor superfamily member, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement regulatory protein be able to.

In some embodiments, the antibody is a humanized CD70 antibody (see, eg, US2009 / 0148942), a humanized CD19 antibody (see, eg, US2009 / 0136526), a chimeric or humanized CD30 antibody (eg, , US2010 / 0239571), humanized CD33 antibody (US2013 / 0309223), humanized beta6 antibody (see eg WO2013 / 123152), or humanized Liv-1 antibody (eg US2013 / 0259860). See).
Drug load-"p"

  Referring again generally to the ligand-linker-drug conjugates of Formulas I, I 'and II, the number of drug-linker units per ligand is represented by p. (In this context, the drug-linker drug may be a cytotoxic agent.) In embodiments where the linker is not branched, p represents the number of drug-linker molecules per ligand (eg, antibody). When referring to individual conjugates, p is an integer representing the number of drug-linker molecules per ligand. When referring to a composition containing multiple conjugates, p is the average number of drug-linkers per ligand (or drug-linker per ligand (eg, antibody) in embodiments where the linker is not branched). The average number of molecules). The variable p ranges from 4 to 20, typically 6 to 12, 8 to 12, or 8 to 16, or 20.

The average number of drug-linker units per ligand unit in preparation from the conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay, HIC and HPLC. The quantitative distribution with respect to p of the ligand-linker-drug conjugate can also be determined. In some cases, separation, purification, and characterization of homogeneous ligand-drug conjugates (p is a specific value from ligand-drug conjugates with other drug loadings) can be accomplished by means such as reverse phase It can be achieved by HPLC or electrophoresis.
Activity assay

There are a number of different assays that can be used to determine whether a ligand-drug conjugate exerts a cytotoxic effect on a cell line. In one example for determining whether a ligand-drug conjugate exerts a cytotoxic effect on a cell line, a thymidine incorporation assay is used. For example, cells at a density of 5,000 cells / 96-well plate are cultured for 72 hours and exposed to 0.5 μCi of ³ H-thymidine during the last 8 hours of the 72 hour period. Incorporation of ³ H-thymidine into the cells of the culture is measured in the presence and absence of the conjugate. The ligand-drug conjugate was cultured under the same conditions, but when the cells of the culture reduced ³ H-thymidine incorporation compared to the same cell that was not contacted with the ligand-drug conjugate, Has a cytotoxic effect on (See also Klussman et al., Bioconjugate Chemistry, 15: 765-773 (2004); Doronina et al., Bioconjugate Chemistry, 17: 114-124 (2006)).

In another example, to determine whether a ligand-drug conjugate exerts a cytotoxic effect on a cell line, cell viability is measured in a cell by a dye, such as neutral red, trypan blue, or ALAMAR ( (Trade Mark) Measured by determining blue uptake (see, eg, Page et al., 1993, Intl. J. of Oncology, 3: 473-476). In such an assay, the cells are incubated in a medium containing the dye, the cells are washed, and the remaining dye reflecting the cellular uptake of the dye is measured spectrophotometrically. The protein-bound dye sulforhodamine B (SRB) can also be used to measure cytotoxicity (Skehan et al., 1990, J. Nat'l Cancer Inst., 82: 1107-12). Preferred ligand-drug conjugates have an IC ₅₀ value of less than 1000 ng / ml, preferably less than 500 ng / ml, more preferably less than 100 ng / ml, even more preferably less than 50 or even less than 10 ng / ml for cell lines ( Including those having a defined mAB concentration that produces 50% cell death.
Drug-linker compound

In another aspect, the formula
A drug-linker compound having the formula: or a pharmaceutically acceptable salt or solvate thereof,
Where
L ^A is a ligand binding component,
L ^H is a hydrophilic linker that is optionally branched, each branch having the formula -AA ₁ -R ^L1 -R ^L2 -R ^L3-
Where
AA ₁ is a hydrophilic amino acid that forms a cleavable bond with the C-terminus of the _DE unit to which it is attached;
R ^L1 is optional, and when R ^L1 is absent exists vital R ^L2 and R ^L3, alkylene substituted with a hydrophilic amino acid or optionally may share L ^A and atoms,
R ^L2 is optional, and when R ^L2 is not present there vital R ^L3, are selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
R ^L3 is optional, and when R ^L3 is present, is selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
The subscript p ′ is an integer from 1 to 4,
_DE is the effector moiety (as described herein)
The ligand-linker-drug conjugate formed with the drug-linker has a hydrophilicity index that is equal to or less than 2;
Left and right lines L ^H, respectively, indicates the covalent attachment to _{D E} units and ^{L A.}

In a related embodiment IV ′, the following formula
A drug-linker compound having the formula: or a pharmaceutically acceptable salt or solvate thereof,
Where
L ^A is a ligand binding component,
L ^H represents the formula -AA ₁ -R ^L1 -R ^L2 -R ^L3-
A hydrophilic linker that is optionally branched with
Where
AA ₁ is a hydrophilic amino acid that forms a cleavable bond with the C-terminus of the _DE unit to which it is attached;
When R ^L3 and the R ^L3 is absent, R ^L1 is alkylene substituted with a hydrophilic amino acid or optionally may share L ^A and atoms,
R ^L2 is optional, and when R ^L2 is not present there vital R ^L3, are selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
R ^L3 is optional, and when R ^L3 is present, is selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
L ^A is a ligand binding component,
The subscript p ′ is an integer from 1 to 4,
_DE is the effector moiety (as described herein)
The ligand-linker-drug conjugate formed from the drug-linker has a hydrophilicity index that is equal to or less than 2;
Left and right lines L ^H, respectively, indicates the covalent attachment to _{D E} units and ^{L A.}

Various components of the ligand-linker-drug conjugates of formulas IV and IV ′ are provided in more detail below.
Drug unit, D _E

Referring to Formulas IV and IV ′, the drug unit, _DE, is represented by the formula
An effector portion having
Where
Each of R ¹ and R ² is independently selected from the group consisting of hydrogen (H) and optionally substituted —C ₁ -C ₈ alkyl, provided that both R ³ and R ^{3 ′} are not H Except in cases, R ¹ and R ² are not both H,
R ³ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
R ^{3 ′} is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
At least one of R ³ and R ^{3 ′} is not H,
R ⁴ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
R ⁵ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
Or R ⁴ and R ⁵ together form a carbocyclic ring and the formula — (CR ^a R ^b ) _n — (wherein R ^a and R ^b are H and optionally substituted —C Independently selected from the group consisting of _{1 to} C ₈ alkyl, and n is selected from the group consisting of 2, 3, 4, 5 and 6.
R ⁶ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
R ⁷ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
Each R ⁸ is independently from the group consisting of H, —OH, optionally substituted —C ₁ -C ₈ alkyl, and optionally substituted —O— (C ₁ -C ₈ alkyl). Selected
R ¹² is H, optionally substituted —C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted —X ¹ aryl, optionally substituted— C ₃ ~C ₈ ^-X 1 in which carbocyclic ring, optionally substituted with - _(C 3 ~C ₈ carbocycle), _-C is optionally substituted 1 -C ₈ alkylene -NH _2, optionally Selected from substituted -C ₃ -C ₈ heterocycle and optionally substituted -X ^1- (C ₃ -C ₈ heterocycle);
Each X ¹ is independently -C ₁ -C ₁₀ alkylene.

In some related embodiments, R ¹² is not a side chain of phenylalanine or a side chain of proline. In some further related embodiments, R ¹² is not a phenylalanine side chain, a methionine side chain, a tryptophan side chain, or a proline side chain.

In some embodiments, R ¹² is selected from natural L-amino acids (other than proline) and the side chain of glycine. In some further embodiments, R ¹² is selected from the side chains of natural L-amino acids other than proline, glycine and phenylalanine. In some further embodiments, R ¹² is selected from the side chains of natural L-amino acids other than proline, glycine, tryptophan, methionine and phenylalanine.

In some further embodiments, R ¹² is threonine, serine, asparagine, aspartic acid, glutamine, glutamic acid, homoserine, hydroxyvaline, furylalanine, threonine (PO ₃ H ₂ ), pyrazolylalanine, triazolylalanine and thiayl. Selected from the side chain of the group of hydrophilic amino acids consisting of zolylalanine.

In some embodiments, R ¹² is the threonine side chain.
Linker unit

Referring to the linker unit of the formula IV and IV ', in some embodiments, L ^A is covalently linked to the sulfur atom of the ligand. In some embodiments, the sulfur atom is a sulfur atom of a cysteine residue that forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is the sulfur atom of a cysteine residue introduced into the ligand unit (eg, by site-directed mutagenesis or a chemical reaction). In a further embodiment, a sulfur atom L ^A is attached is cysteine residues forming the interchain disulfide bonds of the antibody, and were introduced into the Ligand unit (e.g., by site-directed mutagenesis or chemical reaction) cysteine Selected from residues.

AA ₁ forms a cleavable bond with an effector moiety, D _E , eg, a drug unit. In embodiments where AA ₁ is bound to an amino acid of _{D E,} AA ₁ forms a cleavable peptide bond with the _{D E.} A cleavable peptide bond is susceptible to cleavage by a protease when the conjugate reaches its target site. In other embodiments, AA ₁ forms an amide bond with the binding site of the effector moiety that is susceptible to cleavage (eg, by a protease) when the conjugate reaches its targeted site. In some embodiments, AA ₁ is selected from the group consisting of hydrophilic amino acids, typically glycine, and the L form of aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine. Is a natural amino acid. In some embodiments, AA ₁ is glutamate.

In embodiments where R ^L1 is present and is a hydrophilic amino acid, it is glycine; aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine in L or D form; —NH—CH ( R ^a ) —C (O) —; and —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , _{-CH 2 CH 2 CH 2 NH 2} , -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, and _-CH 2 _CH 2 _CH 2 CH ₂ is selected from CO ₂ H, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH ₂ NH—, —CH ₂ CH ₂ C (O) —, —CH ₂ CH ₂ CH ₂ C (O) —, and —CH ₂ CH ₂ CH ₂ CH ₂ C (O) —. In some further embodiments, R ^L1 is D-amino acid of aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; glycine; —NH—CH (R ^a ) —C (O) — And selected from the group consisting of —NH—CH (COOH) —R ^b —, wherein R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ OH, —CH ₂ CH ₂ CH ₂ CO ₂ H, and —CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H, wherein R ^b is — _{CH 2 NH -, - CH 2} CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 C (O _{-, - CH 2 CH 2 CH} 2 C (O) -, and _{-CH 2 CH 2 CH 2 CH 2} C (O) - is selected from.

In embodiments wherein R ^L1 is present and is optionally substituted alkylene, it is —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl) —, — It may be C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents selected from NH ₂ or —NH (C ₁ -C ₃ alkyl). In some embodiments, R ^L1 is ethylenediamine, —NH—CH (COOH) —CH ₂ —NH—, or —C (O) —CH (CH ₂ NH ₂ ) —.

In embodiments where R ^L2 is present and is a hydrophilic amino acid, it is glycine; aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine in the L or D form; —NH—CH ( R ^a ) —C (O) —; and —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , _{-CH 2 CH 2 CH 2 NH 2} , -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, and _-CH 2 _CH 2 _CH 2 CH ₂ is selected from CO ₂ H, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH ₂ NH—, —CH ₂ CH ₂ C (O) —, —CH ₂ CH ₂ CH ₂ C (O) —, and —CH ₂ CH ₂ CH ₂ CH ₂ C (O) —.

In some further embodiments, when R ^L2 is present, it is an aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine D amino acid; glycine; —NH—CH (R ^a ) — C (O) —; and —NH—CH (COOH) —R ^b —, wherein R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH _{2. NH 2, -CH 2 CH 2 CH} 2 CH 2 NH 2, is selected from _{-CH 2 CH 2 OH, -CH 2} CH 2 CH 2 CO 2 H, and _{-CH 2 CH 2 CH 2 CH 2} CO 2 H, R ^b _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - _{H 2 CH 2 C (O)} -, - CH 2 CH 2 CH 2 C (O) -, and _{-CH 2 CH 2 CH 2 CH 2} C (O) - is selected from.

In embodiments wherein R ^L2 is present and is optionally substituted alkylene, it is —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl) —, — It may be C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents selected from NH ₂ or —NH (C ₁ -C ₃ alkyl). In some embodiments, R ^L2 is ethylenediamine, —NH—CH (COOH) —CH ₂ —NH—, or —C (O) —CH (CH ₂ NH ₂ ) —.

In embodiments where R ^L3 is present and is a hydrophilic amino acid, it is glycine; aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine, L-form or D-form; R ^a ) —C (O) —; and —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , _{-CH 2 CH 2 CH 2 NH 2} , -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, and _-CH 2 _CH 2 _CH 2 CH ₂ is selected from CO ₂ H, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH ₂ NH—, —CH ₂ CH ₂ C (O) —, —CH ₂ CH ₂ CH ₂ C (O) —, and —CH ₂ CH ₂ CH ₂ CH ₂ C (O) —. In some further embodiments, when R ^L3 is present, it is a D amino acid of aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; glycine; —NH—CH (R ^a ) — C (O) —; and —NH—CH (COOH) —R ^b —, wherein R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH _{2. NH 2, -CH 2 CH 2 CH} 2 CH 2 NH 2, is selected from _{-CH 2 CH 2 OH, -CH 2} CH 2 CH 2 CO 2 H, and _{-CH 2 CH 2 CH 2 CH 2} CO 2 H, R ^b _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - _{H 2 CH 2 C (O)} -, - CH 2 CH 2 CH 2 C (O) -, and _{-CH 2 CH 2 CH 2 CH 2} C (O) - is selected from.

In embodiments where R ^L3 is present and is optionally substituted alkylene, it is —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl) —, —NH. It may be C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents selected from ₂ or —NH (C ₁ -C ₃ alkyl). In some embodiments, R ^L3 is ethylenediamine, —NH—CH (COOH) —CH ₂ —NH—, or —C (O) —CH (CH ₂ NH ₂ ) —.

In some embodiments above, AA ₁ is present and R ^L1 , R ^L2 and R ^L3 are absent.

In some embodiments above, AA ₁ is present, R ^L1 is present, and R ^L2 and R ^L3 are absent.

In some embodiments above, AA ₁ is present, R ^L1 is present, R ^L2 is present, and R ^L3 is absent.

In some embodiments above, AA ₁ is present, R ^L1 is present, R ^L2 is present, and R ^L3 is present.

In some embodiments above, AA ₁ is a hydrophilic amino acid, at least one of R ^L1 , R ^L2 and R ^L3 is present and optionally substituted as described above. Alkylene.

In some embodiments above, AA ₁ is glutamate, at least one of R ^L1 , R ^L2, and R ^L3 is present and is optionally substituted alkylene as described above It is.

In some embodiments above, AA ₁ is glutamate, R ^L1 is a hydrophilic amino acid, at least one of R ^L2 and R ^L3 is present, and any as described above Alkylene is optionally substituted.

In some embodiments above, AA ₁ and R ^L1 are hydrophilic amino acids, at least one of R ^L2 and R ^L3 is present and optionally substituted as described above. Alkylene.

In some embodiments above, AA ₁ is a hydrophilic amino acid and R ^L1 and optionally R ^L2 are optionally substituted alkylene as described above.

In some embodiments of the, ^{L H} is glycine dipeptide (Gly-Gly) is also a tripeptide is also not included tetrapeptide. In some embodiments, L ^H does not include the peptide Asn- (D) Lys.

In some embodiments, L ^H comprises a modified peptide having 2-4 amino acids. The modified peptide has, at position 1, an amino acid (AA ₁ ) that is selected to optimize the release of the _DE unit (eg, by protease cleavage via an amide peptide bond). In one or both positions, R ^L1 and R ^L2 reverse the typical N to C linkage orientation of the peptide and promote the binding of the last amino acid (eg, R ^L2 or R ^L3 ). Which contains an α-amino group protected as a maleimide prior to attachment of the ligand unit. An amino acid having an inverted N to C linkage is linked to the next group through its side chain. In some embodiments, the amino acid is an alpha amino acid. In other embodiments, it can be a beta or gamma amino acid. In some of these embodiments, the side chain is —CH ₂ NH ₂ —, —CH ₂ CH ₂ NH ₂ —, —CH ₂ CH ₂ CH ₂ NH ₂ —, and —CH ₂ CH ₂ CH ₂ CH _2. NH ₂ - is selected from.

In some embodiments of L ^H , according to any of the above embodiments, the amino acid having an inverted N to C linkage (R ^L1 ) is bound to R ^L2 or R ^L3 , and R ^L2 Or R ^L3 is a hydrophilic amino acid or an optionally substituted alkylene.

In some embodiments of L ^H, by any of the above embodiments, the amino acid having a connection to C from reversed the N (R ^L1) is attached to R ^L2, R ^L2 is optionally Alkylene which is optionally substituted.

In some further embodiments, L ^H is a hydrophilic cleavable linker and each branch is of the formula
In which R ²¹ is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, —CH ₂ CH ₂ OH, —CH ₂ CO ₂ H, —CH ₂ CH ₂ CO ₂ H, —CH ₂ CH ₂ CH ₂ CO ₂ H, and —CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H, R ²² is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , — CH ₂ OH, is selected from _-CH 2 _CH 2 OH. Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In a further embodiment, L ^H or a branch thereof is of the formula
Wherein R ²² is selected from —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, and —CH ₂ CH ₂ OH. In some embodiments, R ²² is selected from —CH ₂ NH ₂ and —CH ₂ CH ₂ NH ₂ . Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In some further embodiments above, L ^H is of the formula
A branched hydrophilic linker having
In the formula, each R ³¹ is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, —CH ₂ CH ₂ OH, —CH ₂ CO ₂ H, —CH ₂ CH ₂ CO ₂ H, Independently selected from the group consisting of —CH ₂ CH ₂ CH ₂ CO ₂ H and —CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H, each bar represents a binding site for the _DE unit.

In some further embodiments above, L ^H is of the formula
Where each bar represents a bond to a _DE unit.

The L ^A subunit, described in more detail above.

Referring back to the drug linker conjugates of formula IV and IV ′, in some further embodiments, the drug-linker is:
Having an expression selected from:

Certain embodiments are described below.
Or a pharmaceutically acceptable salt or solvate thereof.
Linker

In another embodiment, the formula _{^{(L H) p '-L A}} (VII)
Or a pharmaceutically acceptable salt or solvate thereof, wherein:
L ^H represents the formula -AA ₁ -R ^L1 -R ^L2 -R ^L3-
A hydrophilic linker that is optionally branched with
L ^A is a ligand binding component,
p ′ is an integer of 1 to 4,
Left and right lines L ^H, respectively, indicating the binding sites for _{D E} units and ^{L A.}

Referring to formula VII, in some embodiments, L ^A is covalently linked to the sulfur atom of the ligand. L ^A can be, for example, a maleimide or succinimide that is suitable for bonding to the sulfur atom (succimimide).

AA ₁ can form a cleavable bond with an effector moiety D _E , eg, a drug unit. In embodiments where AA ₁ is bound to an amino acid of _{D E,} AA ₁ forms a cleavable peptide bond with the _{D E.} A cleavable peptide bond is susceptible to cleavage by a protease when the conjugate reaches its target site. In other embodiments, AA ₁ forms an amide bond with the binding site of the effector moiety that is susceptible to cleavage (eg, by a protease) when the conjugate reaches its target site. In some embodiments, AA ₁ is selected from the group consisting of hydrophilic amino acids, typically glycine, and the L form of aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine. Is a natural amino acid. In some embodiments, AA ₁ is glutamate.

In embodiments where R ^L1 is present and is a hydrophilic amino acid, it is glycine; aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine in L or D form; —NH—CH ( R ^a ) —C (O) —; and —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , _{-CH 2 CH 2 CH 2 NH 2} , -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, and _-CH 2 _CH 2 _CH 2 CH ₂ is selected from CO ₂ H, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH ₂ NH—, —CH ₂ CH ₂ C (O) —, —CH ₂ CH ₂ CH ₂ C (O) —, and —CH ₂ CH ₂ CH ₂ CH ₂ C (O) —. In some further embodiments, R ^L1 is D-amino acid of aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; glycine; —NH—CH (R ^a ) —C (O) — And selected from the group consisting of —NH—CH (COOH) —R ^b —, wherein R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ OH, —CH ₂ CH ₂ CH ₂ CO ₂ H, and —CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H, wherein R ^b is — _{CH 2 NH -, - CH 2} CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 C (O _{-, - CH 2 CH 2 CH} 2 C (O) -, and _{-CH 2 CH 2 CH 2 CH 2} C (O) - is selected from.

In embodiments where R ^L1 is present and is optionally substituted alkylene, it is —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl), —NH It may be C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents selected from ₂ or —NH (C ₁ -C ₃ alkyl). In some embodiments, R ^L1 is ethylenediamine, —NH—CH (COOH) —CH ₂ —NH—, or —C (O) —CH (CH ₂ NH ₂ ) —.

In embodiments where R ^L2 is present and is a hydrophilic amino acid, it is glycine; aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine in the L or D form; —NH—CH ( R ^a ) —C (O) —; and —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , _{-CH 2 CH 2 CH 2 NH 2} , -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, and _-CH 2 _CH 2 _CH 2 CH ₂ is selected from CO ₂ H, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH ₂ NH—, —CH ₂ CH ₂ C (O) —, —CH ₂ CH ₂ CH ₂ C (O) —, and —CH ₂ CH ₂ CH ₂ CH ₂ C (O) —. In some further embodiments, when R ^L2 is present, it is an aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine D amino acid; glycine; —NH—CH (R ^a ) — C (O) —; and —NH—CH (COOH) —R ^b —, wherein R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH _{2. NH 2, -CH 2 CH 2 CH} 2 CH 2 NH 2, is selected from _{-CH 2 CH 2 OH, -CH 2} CH 2 CH 2 CO 2 H, and _{-CH 2 CH 2 CH 2 CH 2} CO 2 H, R ^b _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - _{H 2 CH 2 C (O)} -, - CH 2 CH 2 CH 2 C (O) -, and _{-CH 2 CH 2 CH 2 CH 2} C (O) - is selected from.

In embodiments wherein R ^L2 is present and is optionally substituted alkylene, it is —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl) —, — It may be C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents selected from NH ₂ or —NH (C ₁ -C ₃ alkyl). In some embodiments, R ^L2 is ethylenediamine, —NH—CH (COOH) —CH ₂ —NH—, or —C (O) —CH (CH ₂ NH ₂ ) —.

In embodiments where R ^L3 is present and is a hydrophilic amino acid, it is glycine; aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine, L-form or D-form; R ^a ) —C (O) —; and —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , _{-CH 2 CH 2 CH 2 NH 2} , -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, and _-CH 2 _CH 2 _CH 2 CH ₂ is selected from CO ₂ H, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH ₂ NH—, —CH ₂ CH ₂ C (O) —, —CH ₂ CH ₂ CH ₂ C (O) —, and —CH ₂ CH ₂ CH ₂ CH ₂ C (O) —.

In some further embodiments, when R ^L3 is present, it is a D amino acid of aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; glycine; —NH—CH (R ^a ) — C (O) —; and —NH—CH (COOH) —R ^b —, wherein R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH _{2. NH 2, -CH 2 CH 2 CH} 2 CH 2 NH 2, is selected from _{-CH 2 CH 2 OH, -CH 2} CH 2 CH 2 CO 2 H, and _{-CH 2 CH 2 CH 2 CH 2} CO 2 H, R ^b _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - _{H 2 CH 2 C (O)} -, - CH 2 CH 2 CH 2 C (O) -, and _{-CH 2 CH 2 CH 2 CH 2} C (O) - is selected from.

In embodiments where R ^L3 is present and is optionally substituted alkylene, it is —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl) —, —NH. It may be C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents selected from ₂ or —NH (C ₁ -C ₃ alkyl). In some embodiments, R ^L3 is ethylenediamine, —NH—CH (COOH) —CH ₂ —NH—, or —C (O) —CH (CH ₂ NH ₂ ) —.

In some embodiments above, AA ₁ is present and R ^L1 , R ^L2 and R ^L3 are absent.

In some embodiments above, AA ₁ is present, R ^L1 is present, and R ^L2 and R ^L3 are absent.

In some embodiments above, AA ₁ is present, R ^L1 is present, R ^L2 is present, and R ^L3 is absent.

In some embodiments above, AA ₁ is present, R ^L1 is present, R ^L2 is present, and R ^L3 is present.

In some embodiments above, AA ₁ is a hydrophilic amino acid, at least one of R ^L1 , R ^L2 and R ^L3 is present and optionally substituted as described above. Alkylene.

In some embodiments above, AA ₁ is glutamate, at least one of R ^L1 , R ^L2, and R ^L3 is present and is optionally substituted alkylene as described above It is.

In some embodiments above, AA ₁ is glutamate, R ^L1 is a hydrophilic amino acid, at least one of R ^L2 and R ^L3 is present, and any as described above Alkylene is optionally substituted.

In some embodiments above, AA ₁ and R ^L1 are hydrophilic amino acids, at least one of R ^L2 and R ^L3 is present and optionally substituted as described above. Alkylene.

In some embodiments above, AA ₁ is a hydrophilic amino acid and R ^L1 and optionally R ^L2 are optionally substituted alkylene as described above.

In some embodiments of the, ^{L H} is glycine dipeptide (Gly-Gly) is also a tripeptide is also not included tetrapeptide. In some embodiments, L ^H does not include the peptide Asn- (D) Lys.

In some embodiments, L ^H comprises a modified peptide having 2-4 amino acids. The modified peptide has, at position 1, an amino acid (AA ₁ ) that is selected to optimize the release of the _DE unit (eg, by protease cleavage via an amide peptide bond). In one or both positions, R ^L1 and R ^L2 reverse the typical N to C linkage orientation of the peptide and promote the binding of the last amino acid (eg, R ^L2 or R ^L3 ). Which contains an α-amino group protected as a maleimide prior to attachment of the ligand unit. An amino acid having an inverted N to C linkage is linked to the next group through its side chain. In some embodiments, the amino acid is an alpha amino acid. In other embodiments, it can be a beta or gamma amino acid. In some of these embodiments, the side chain is —CH ₂ NH ₂ —, —CH ₂ CH ₂ NH ₂ —, —CH ₂ CH ₂ CH ₂ NH ₂ —, and —CH ₂ CH ₂ CH ₂ CH _2. NH ₂ - is selected from.

In some embodiments of L ^H , according to any of the above embodiments, the amino acid having an inverted N to C linkage (R ^L1 ) is bound to R ^L2 or R ^L3 , and R ^L2 Or R ^L3 is a hydrophilic amino acid or an optionally substituted alkylene.

In some embodiments of L ^H, by any of the above embodiments, the amino acid having a connection to C from reversed the N (R ^L1) is attached to R ^L2, R ^L2 is optionally Alkylene which is optionally substituted.

In some further embodiments, L ^H is a hydrophilic cleavable linker and each branch is of the formula
In which R ²¹ is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, —CH ₂ CH ₂ OH, —CH ₂ CO ₂ H, —CH ₂ CH ₂ CO ₂ H, —CH ₂ CH ₂ CH ₂ CO ₂ H, and —CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H, R ²² is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , — CH ₂ OH, is selected from _-CH 2 _CH 2 OH. Left and right wavy line respectively indicate a binding site for D _E units and L ^A.

In a further embodiment, L ^H or a branch thereof is of the formula
Wherein R ²² is selected from —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, and —CH ₂ CH ₂ OH. In some embodiments, R ²² is selected from —CH ₂ NH ₂ and —CH ₂ CH ₂ NH ₂ . Left and right wavy line respectively indicate a binding site for D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate a binding site for D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate a binding site for D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate a binding site for D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate a binding site for D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate a binding site for D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate a binding site for D _E units and L ^A.

In some further embodiments above, L ^H is of the formula
Wherein each R ³¹ is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, —CH ₂ CH ₂ OH, —CH ₂ CO Each independently selected from the group consisting of ₂ H, —CH ₂ CH ₂ CO ₂ H, —CH ₂ CH ₂ CH ₂ CO ₂ H, and —CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H, Denotes the binding site for the _DE unit.

In some further embodiments above, L ^H is of the formula
Where each bar represents a binding site for the _DE unit and a vertical dashed line represents the binding site for the ligand unit.

The L ^A subunit, described in more detail herein.

In each of the embodiments shown above, each of the amine, hydroxyl and carboxylic acid groups is in an optionally protected form. Suitable protecting groups are described, for example, in Greene and Wuts, Protective Groups in Organic Synthesis, J. MoI. Provided in Wiley & Sons and generally selected to be removable independently of each other.
Linker-ligand conjugate

In yet another aspect,
_{^{([L H] p '-L}} A) p -L (X)
A linker-ligand conjugate having a formula selected from: or a pharmaceutically acceptable salt or solvate thereof,
Where
L is a ligand that specifically binds to the target;
L ^H is a hydrophilic linker that is optionally branched, and each branch of L ^H has the formula -AA ₁ -R ^L1 -R ^L2 -R ^L3-
Have
L ^A is a ligand binding component,
The subscript p is an integer of about 4 to 20,
The subscript p ′ is an integer from 1 to 4,
Left and right lines L ^H, respectively, indicates the binding site on the _{D E} units and ^{L A.}

AA ₁ can form a cleavable bond with an effector moiety D _E , eg, a drug unit. In embodiments where AA ₁ is bound to an amino acid of _{D E,} AA ₁ forms a cleavable peptide bond with the _{D E.} A cleavable peptide bond is susceptible to cleavage by a protease when the conjugate reaches its target site. In other embodiments, AA ₁ forms an amide bond with the binding site of the effector moiety that is susceptible to cleavage (eg, by a protease) when the conjugate reaches its target site. In some embodiments, AA ₁ is selected from the group consisting of hydrophilic amino acids, typically glycine, and the L form of aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine. Is a natural amino acid. In some embodiments, AA ₁ is glutamate.

In some embodiments, L ^A is covalently linked to the sulfur atom of the ligand. L ^A can be, for example, a maleimide or succinimide that is suitable for bonding to a sulfur atom.

In embodiments where R ^L1 is present and is a hydrophilic amino acid, it is glycine; aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine in L or D form; —NH—CH ( R ^a ) —C (O) —; and —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , _{-CH 2 CH 2 CH 2 NH 2} , -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, and _-CH 2 _CH 2 _CH 2 CH ₂ is selected from CO ₂ H, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH ₂ NH—, —CH ₂ CH ₂ C (O) —, —CH ₂ CH ₂ CH ₂ C (O) —, and —CH ₂ CH ₂ CH ₂ CH ₂ C (O) —. In some further embodiments, R ^L1 is D-amino acid of aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine; glycine; —NH—CH (R ^a ) —C (O) — And selected from the group consisting of —NH—CH (COOH) —R ^b —, wherein R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ OH, —CH ₂ CH ₂ CH ₂ CO ₂ H, and —CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H, wherein R ^b is — _{CH 2 NH -, - CH 2} CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 C (O _{-, - CH 2 CH 2 CH} 2 C (O) -, and _{-CH 2 CH 2 CH 2 CH 2} C (O) - is selected from.

In embodiments wherein R ^L1 is present and is optionally substituted alkylene, it is —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl) —, — It may be C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents selected from NH ₂ or —NH (C ₁ -C ₃ alkyl). In some embodiments, R ^L1 is ethylenediamine, —NH—CH (COOH) —CH ₂ —NH—, or —C (O) —CH (CH ₂ NH ₂ ) —.

In embodiments where R ^L2 is present and is a hydrophilic amino acid, it is glycine; aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine, and alanine in the L or D form; —NH—CH ( R ^a ) —C (O) —; and —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , _{-CH 2 CH 2 CH 2 NH 2} , -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, and _-CH 2 _CH 2 _CH 2 CH ₂ is selected from CO ₂ H, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH ₂ NH—, —CH ₂ CH ₂ C (O) —, —CH ₂ CH ₂ CH ₂ C (O) —, and —CH ₂ CH ₂ CH ₂ CH ₂ C (O) —.

In some further embodiments, when R ^L2 is present, it is an aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine D amino acid; glycine; —NH—CH (R ^a ) — C (O) —; and —NH—CH (COOH) —R ^b —, wherein R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH _{2. NH 2, -CH 2 CH 2 CH} 2 CH 2 NH 2, is selected from _{-CH 2 CH 2 OH, -CH 2} CH 2 CH 2 CO 2 H, and _{-CH 2 CH 2 CH 2 CH 2} CO 2 H, R ^b _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - _{H 2 CH 2 C (O)} -, - CH 2 CH 2 CH 2 C (O) -, and _{-CH 2 CH 2 CH 2 CH 2} C (O) - is selected from.

In embodiments wherein R ^L2 is present and is optionally substituted alkylene, it is —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl) —, — It may be C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents selected from NH ₂ or —NH (C ₁ -C ₃ alkyl). In some embodiments, R ^L2 is ethylenediamine, —NH—CH (COOH) —CH ₂ —NH—, or —C (O) —CH (CH ₂ NH ₂ ) —.

In embodiments where R ^L3 is present and is a hydrophilic amino acid, it is glycine; aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine, L-form or D-form; R ^a ) —C (O) —; and —NH—CH (COOH) —R ^b —, where R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , _{-CH 2 CH 2 CH 2 NH 2} , -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CO 2 H, and _-CH 2 _CH 2 _CH 2 CH ₂ is selected from CO ₂ H, ^{R b} _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH ₂ NH—, —CH ₂ CH ₂ C (O) —, —CH ₂ CH ₂ CH ₂ C (O) —, and —CH ₂ CH ₂ CH ₂ CH ₂ C (O) —. In some further embodiments, when R ^L3 is present, aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine D amino acids; glycine; —NH—CH (R ^a ) —C ( O) —; and —NH—CH (COOH) —R ^b —, wherein R ^a is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ CH ₂ CH ₂ NH _{2. , -CH 2 CH 2 CH 2 CH} 2 NH 2, is selected from _{-CH 2 CH 2 OH, -CH 2} CH 2 CH 2 CO 2 H, and _{-CH 2 CH 2 CH 2 CH 2} CO 2 H, R b _{is, -CH 2 NH -, - CH} 2 CH 2 NH -, - CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - CH 2 C _{H 2 C (O) -,} - CH 2 CH 2 CH 2 C (O) -, and _{-CH 2 CH 2 CH 2 CH 2} C (O) - is selected from.

In embodiments where R ^L3 is present and is optionally substituted alkylene, it is —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl) —, —NH. It may be C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents selected from ₂ or —NH (C ₁ -C ₃ alkyl). In some embodiments, R ^L3 is ethylenediamine, —NH—CH (COOH) —CH ₂ —NH—, or —C (O) —CH (CH ₂ NH ₂ ) —.

In some embodiments above, AA ₁ is present and R ^L1 , R ^L2 and R ^L3 are absent.

In some embodiments above, AA ₁ is present, R ^L1 is present, and R ^L2 and R ^L3 are absent.

In some embodiments above, AA ₁ is present, R ^L1 is present, R ^L2 is present, and R ^L3 is absent.

In some embodiments above, AA ₁ is present, R ^L1 is present, R ^L2 is present, and R ^L3 is present.

In some embodiments above, AA ₁ is a hydrophilic amino acid, at least one of R ^L1 , R ^L2 and R ^L3 is present and optionally substituted as described above. Alkylene.

In some embodiments above, AA ₁ is glutamate, at least one of R ^L1 , R ^L2, and R ^L3 is present and is optionally substituted alkylene as described above It is.

In some embodiments above, AA ₁ is glutamate, R ^L1 is a hydrophilic amino acid, at least one of R ^L2 and R ^L3 is present, and any as described above Alkylene is optionally substituted.

In some embodiments above, AA ₁ and R ^L1 are hydrophilic amino acids, at least one of R ^L2 and R ^L3 is present and optionally substituted as described above. Alkylene.

In some embodiments above, AA ₁ is a hydrophilic amino acid and R ^L1 and optionally R ^L2 are optionally substituted alkylene as described above.

In some embodiments of the, ^{L H} is glycine dipeptide (Gly-Gly) is also a tripeptide is also not included tetrapeptide. In some embodiments, L ^H does not include the peptide Asn- (D) Lys.

In some embodiments, L ^H comprises a modified peptide having 2-4 amino acids. The modified peptide has, at position 1, an amino acid (AA ₁ ) that is selected to optimize the release of the _DE unit (eg, by protease cleavage via an amide peptide bond). In one or both positions, R ^L1 and R ^L2 reverse the typical N to C linkage orientation of the peptide and promote the binding of the last amino acid (eg, R ^L2 or R ^L3 ). Which contains an α-amino group protected as a maleimide prior to attachment of the ligand unit. An amino acid having an inverted N to C linkage is linked to the next group through its side chain. In some embodiments, the amino acid is an alpha amino acid. In other embodiments, it can be a beta or gamma amino acid. In some of these embodiments, the side chain is —CH ₂ NH ₂ —, —CH ₂ CH ₂ NH ₂ —, —CH ₂ CH ₂ CH ₂ NH ₂ —, and —CH ₂ CH ₂ CH ₂ CH _2. NH ₂ - is selected from.

In some embodiments of L ^H , according to any of the above embodiments, the amino acid having an inverted N to C linkage (R ^L1 ) is bound to R ^L2 or R ^L3 , and R ^L2 Or R ^L3 is a hydrophilic amino acid or an optionally substituted alkylene.

In some embodiments of L ^H, by any of the above embodiments, the amino acid having a connection to C from reversed the N (R ^L1) is attached to R ^L2, R ^L2 is optionally Alkylene which is optionally substituted.

In some further embodiments, L ^H is a hydrophilic cleavable linker and each branch is of the formula
In which R ²¹ is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, —CH ₂ CH ₂ OH, —CH ₂ CO ₂ H, —CH ₂ CH ₂ CO ₂ H, —CH ₂ CH ₂ CH ₂ CO ₂ H, and —CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H, R ²² is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , — CH ₂ OH, is selected from _-CH 2 _CH 2 OH. Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In a further embodiment, L ^H or a branch thereof is of the formula
Wherein R ²² is selected from —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, and —CH ₂ CH ₂ OH. In some embodiments, R ²² is selected from —CH ₂ NH ₂ and —CH ₂ CH ₂ NH ₂ . Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In certain embodiments, L ^H or a branch thereof is of the formula
Have Left and right wavy line respectively indicate the binding of D _E units and L ^A.

In some further embodiments above, L ^H is of the formula
A branched hydrophilic linker having
In the formula, each R ³¹ is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, —CH ₂ CH ₂ OH, —CH ₂ CO ₂ H, —CH ₂ CH ₂ CO ₂ H, Independently selected from the group consisting of —CH ₂ CH ₂ CH ₂ CO ₂ H, and —CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H, each of the bars represents a binding site for the _DE unit and is a vertical dashed line Indicates the binding site for the ligand unit.

In some further embodiments above, L ^H is of the formula
A branched hydrophilic linker having
In the formula, each of the bars indicates binding to the _DE unit, and the vertical dashed line indicates the binding site for the ligand unit.

The L ^A subunit, described in more detail above.

In each of the embodiments shown above, each of the amine, hydroxyl and carboxylic acid groups is optionally in protected form. Suitable protecting groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, J. MoI. Provided in Wiley & Sons and generally selected to be removable independently of each other.
Where P is H or a protecting group, the remaining functional groups present in R ¹ , R ² , each of the indicated carboxylic acids is optionally protected, and S is the ligand It is a sulfur atom.
Disease treatment

Further provided is a method of treatment of a disease using a ligand-linker-drug conjugate of any of the formulas described herein. The disease can be, for example, cancer or an autoimmune disease. The ligand-linker-drug conjugate is administered in a therapeutically effective amount and a therapeutically effective schedule. In some embodiments, the conjugate dose is the same or less than a comparable two-loaded conjugate dose. In some embodiments, the conjugate dose is the same or less than that of a comparable 4-loaded conjugate. In some embodiments, the conjugate dose is the same or less than a comparable two-loaded conjugate dose, while the dosing schedule is the same or less frequent . In some embodiments, the conjugate dose is the same or less than a comparable 4-loaded conjugate dose, while the dosing schedule is the same or less frequent . In some further embodiments, the conjugate dose is higher and the dosing schedule is the same or less frequent than the dosing schedule of comparable two-loaded conjugates. In some further embodiments, the conjugate dose is lower and the dosing schedule is the same or less frequent than the dosing schedule of a comparable 4-loaded conjugate. The comparative conjugate can be, for example, the same ligand-drug-linker conjugate with 2 or 4 drug loadings.
Cancer treatment

  The ligand-linker-drug conjugate is useful for inhibiting the growth of tumor cells or cancer cells, causing apoptosis in the tumor or cancer cells, or treating cancer in a patient. The ligand-linker-drug conjugate can be used accordingly in various settings for the treatment of cancer. Ligand-drug conjugates can be used to deliver drugs to tumor cells or other cancer cells. Without being bound by theory, in one embodiment, the ligand unit of the ligand-linker-drug conjugate specifically binds to a target (eg, an antigen on a cancer cell) and the ligand-drug conjugate. May be taken up (internalized) into tumor cells or cancer cells by receptor-mediated endocytosis or other internalization mechanisms. The antigen can be bound to the tumor cell or cancer cell or can be an extracellular matrix protein associated with the tumor cell or cancer cell. Immediately inside the cell, the drug (cytotoxic agent) is released inside the cell. In an alternative embodiment, the drug or drug unit is cleaved from the ligand-linker-drug conjugate outside the tumor cell or cancer cell, and the drug or drug unit subsequently penetrates the cell.

  Ligand-linker-drug conjugates can provide conjugation-specific tumor or cancer drug targeting and thus reduce the general toxicity of the drug (when administered alone).

  In one embodiment, the ligand unit specifically binds to a tumor cell or cancer cell.

  In another embodiment, the ligand unit specifically binds to a tumor cell or cancer cell antigen that is on the surface of the tumor cell or cancer cell.

  In another embodiment, the ligand unit specifically binds to a tumor cell or cancer cell antigen that is an extracellular matrix protein associated with the tumor cell or cancer cell.

  The specificity of the ligand unit for a particular tumor cell or cancer cell can be important to determine the most effectively treated tumor or cancer.

  Specific types of cancer that can be treated with a ligand-linker-drug conjugate include, but are not limited to, solid tumors (eg, renal cell cancer, liver cancer and skin cancer) and blood-derived Cancers such as acute myeloblastic leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL) and multiple myeloma are included. Acute and chronic leukemias and lymphomas (eg, Hodgkin lymphoma and non-Hodgkin lymphoma) can be treated.

  Cancers, including but not limited to tumors, metastases, or other diseases or disorders characterized by uncontrolled cell growth, can be treated or inhibited by administration of a ligand-linker drug conjugate.

  In other embodiments, a method of treating cancer is provided that comprises administering to a patient in need thereof an effective amount of a ligand-drug conjugate and a chemotherapeutic agent. In one embodiment, the chemotherapeutic agent is one that has not been found to be refractory to cancer treatment. In another embodiment, the chemotherapeutic agent is one that has been found to be refractory to the treatment of cancer. The ligand-drug conjugate can be administered to patients who also undergo surgery as a treatment for cancer.

  In some embodiments, the patient also receives further treatment, eg, radiation therapy. In certain embodiments, the ligand-drug conjugate is administered concurrently with the chemotherapeutic agent or with radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered or administered before or after administration of the ligand drug conjugate.

  Chemotherapeutic agents can be administered over a series of sessions or as a single dose. Any one or combination of chemotherapeutic agents can be administered, eg, standard treatment chemotherapeutic agent (s).

Furthermore, methods of cancer treatment with ligand-drug conjugates have proven that chemotherapy or radiation therapy is quite toxic to the subject being treated, for example, leading to unacceptable or intolerable side effects. Or as an alternative to chemotherapy or radiation therapy where it can be demonstrated. The patient to be treated can optionally be treated with another cancer treatment, such as surgery, radiation therapy or chemotherapy, depending on which treatment is found to be acceptable or tolerable.
Treatment of autoimmune diseases

  Ligand-drug conjugates cause or kill the cell (s) that cause or participate in the autoimmune disease or inhibit replication of the cell (s) or treat the autoimmune disease Useful for. The ligand-drug conjugate can be used accordingly in various settings for the treatment of autoimmune diseases in patients. Ligand-drug conjugates can be used to deliver drugs to target cells. Without being bound by theory, in one embodiment, the ligand-drug conjugate specifically binds to an antigen on the surface of the target cell, and then the ligand-linker-drug conjugate is receptor-mediated. It is taken up into target cells by endocytosis or other internalization mechanisms. Immediately inside the cell, the drug unit is released. The released drug units then move freely within the cytosol and induce cytotoxic or cytostatic activity. In an alternative embodiment, the drug is cleaved from the ligand-drug conjugate outside the target cell, and the drug or drug unit subsequently penetrates the cell.

  In one embodiment, the ligand unit binds to an autoimmune antigen. In one aspect, the antigen is present on the surface of a cell that is involved in an autoimmune condition.

  In another embodiment, the ligand unit binds to an autoimmune antigen that is on the surface of the cell.

  In one embodiment, the ligand unit binds to activated lymphocytes associated with an autoimmune disease state.

  In further embodiments, the ligand-drug conjugate kills or inhibits the growth of cells that produce autoimmune antibodies associated with certain autoimmune diseases.

  Specific types of autoimmune diseases that can be treated with a ligand drug conjugate include, but are not limited to, disorders involving Th2 lymphocytes (eg, atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergic) Rhinitis, Omen's syndrome, systemic sclerosis, and graft-versus-host disease); disorders associated with Th1 lymphocytes (eg, rheumatoid arthritis, multiple sclerosis, psoriasis, Sjogren's syndrome, Hashimoto's thyroiditis) , Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, and tuberculosis); including disorders associated with activated B lymphocytes (eg, systemic lupus erythematosus, Goodpasture syndrome, rheumatoid arthritis, and type I diabetes) .

A method of treating an autoimmune disease comprising administering to a patient in need thereof an effective amount of a ligand-linker-drug conjugate and another therapeutic agent known for the treatment of autoimmune disease Also disclosed.
Infectious disease treatment

  Ligand-linker-drug conjugates are useful for killing or inhibiting the growth of cells that cause infections or for treating infections. The ligand-linker-drug conjugate can be used accordingly in various settings for the treatment of infections in patients. Ligand-linker-drug conjugates can be used to deliver drugs (eg, antibiotics) to target cells. In one embodiment, the ligand unit binds to the infectious disease cell.

  In one embodiment, the conjugate kills or inhibits the growth of cells that cause a particular infection.

Disclosed is a method of treating an infection comprising administering to a patient in need thereof a ligand-linker-drug conjugate and another therapeutic agent that is an anti-infective agent.
Compositions and methods of administration

  The present invention further provides a pharmaceutical composition comprising a ligand-linker-drug conjugate as described herein and a pharmaceutically acceptable carrier. The ligand-linker-drug conjugate may be in any form that allows the compound to be administered to a patient for the treatment of disorders associated with expression of an antigen to which the ligand unit specifically binds. For example, the conjugate can be in liquid or solid form. The preferred route of administration is parenteral. Parenteral administration includes subcutaneous, intravenous, intramuscular, intrasternal injection or infusion techniques. In one aspect, the composition is administered parenterally. In another aspect, the compound is administered intravenously.

  The pharmaceutical composition of the ligand-linker-drug conjugate can be formulated to allow the compound to be bioavailable for administration of the conjugate to a patient. The composition may take the form of one or more dosage units, for example, a vial may be a single dosage unit.

  The materials used in the preparation of the pharmaceutical composition can be non-toxic in the amounts used. It will be apparent to those skilled in the art that the optimal dosage of active ingredient (s) in a pharmaceutical composition will depend on a variety of factors. Related factors include, but are not limited to, the type of animal (eg, human), the particular form of the compound, the mode of administration, and the composition used.

  The pharmaceutical composition may be, for example, in liquid form. Liquids can be useful for delivery by injection. In a composition for administration by injection or intravenous administration, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffering agent, stabilizer and / or isotonic agent may also be present. Can be included.

  Liquid compositions can also include one or more of the following, whether in solution, suspension, or other similar form. Sterile excipients such as water for injection, saline solution, preferably saline, Ringer's solution, isotonic sodium chloride, polyethylene glycol, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol Or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as amino acids, acetic acid, citric acid or phosphoric acid; detergents such as nonionic surfactants Polyols; and agents for regulating osmotic pressure, such as sodium chloride or dextrose. The parenteral composition can be enclosed in ampoules, disposable syringes or single or multiple dose vials made of glass, plastic or other materials. Saline is an exemplary adjuvant. Injectable compositions are preferably sterile.

  The amount of conjugate effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be used to help identify optimal dosage ranges. The exact dose used in a pharmaceutical composition will also depend on the route of administration and the severity of the disease or disorder and should be determined by the judgment of the physician and the circumstances of each patient.

  The composition includes an amount of the compound so that a suitable dosage is obtained. Typically, this amount is at least about 0.01% compound by weight of the composition.

  For intravenous administration, the composition can comprise from about 0.01 to about 10 mg of ligand-linker-drug conjugate per kg of animal body weight. In one aspect, the composition can comprise about 0.01 to about 10 mg of the ligand-drug conjugate per kg of animal body weight. In another aspect, the amount administered ranges from about 0.1 to about 7.5 mg / kg body weight of the compound.

  In general, the dosage of a compound administered to a patient is typically about 0.01 mg / kg to about 10 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 0.01 mg / kg to about 7.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 0.1 mg / kg to about 5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 0.1 mg / kg to about 4 mg / kg of the subject's body weight. In some embodiments, the dosage administered is about 0.1 mg / kg to about 3 mg / kg or about 0.1 mg / kg to about 2 mg / kg of the subject's body weight. In some embodiments, the dosage administered is about 0.5 mg / kg to about 5 mg / kg of the subject's body weight. In some embodiments, the dosage administered is about 1 mg / kg to about 5 mg / kg of the subject's body weight. In some embodiments, the dose administered is about 0.1 to 4 mg / kg, even more preferably 0.1 to 3.2 mg / kg, or even more preferably 0. 1-2.7 mg / kg body weight of the subject.

  The ligand-linker drug conjugate can be administered by absorption through the epithelial lining or mucocutaneous lining (eg, oral mucosa, rectum and intestinal mucosa) by any convenient route, eg, by infusion or bolus injection. . Administration can be systemic or local. Various delivery systems are known, eg, encapsulation in liposomes, microparticles, microcapsules, capsules, and various delivery systems can be used to administer the compound. In certain embodiments, more than one compound or composition is administered to the patient.

  The term “carrier” refers to an excipient, adjuvant or additive with which the compound is administered. Such a pharmaceutical carrier may be a liquid, for example water. The carrier can be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea. In addition, auxiliaries, stabilizers, thickeners, lubricants and colorants can be used. In one embodiment, when administered to a patient, the compound or composition and the pharmaceutically acceptable carrier are sterile. Water is an exemplary carrier when the compound is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are also additives such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, Contains glycerol, propylene, glycol, water, ethanol. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.

  In one embodiment, the conjugate is formulated by routine procedures as a pharmaceutical composition adapted for intravenous administration to animals, particularly humans. Typically, a carrier or vehicle for intravenous administration is a sterile isotonic aqueous buffer solution. If necessary, the composition can also include a solubilizer. Compositions for intravenous administration can optionally contain a local anesthetic such as lignocaine to ease pain at the site of the injection. In general, the ingredients are mixed separately or in unit dosage form, for example, lyophilized powder or water-free concentrate in a sealed container, for example an ampoule or sachet indicating the amount of active agent Supplied as a product. Where the conjugate is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the conjugate is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

  Pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the US Food and Drug Administration.

The pharmaceutical composition of the present invention comprises the ligand drug conjugate of the present invention and a pharmaceutically acceptable carrier. In some preferred embodiments, all, or substantially all, or more than 50% of the ligand drug conjugate present in the pharmaceutical composition comprises hydrolyzed thio-substituted succinimide. In some preferred embodiments, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, 90% of the ligand drug conjugate present in the pharmaceutical composition More than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% contain hydrolyzed thio-substituted succinimide.
Method for preparing a ligand-drug conjugate

  In another aspect, the present invention provides methods for preparing ligand-drug conjugates, linkers, drug-linkers and linker-ligand conjugates.

  In some embodiments, a method of the invention comprises providing a drug-linker or linker unit as described herein, and conjugating the drug-linker or linker unit to a sulfhydryl group of a ligand unit. Forming a conjugate. In some further embodiments, the thio-substituted maleimide or succinimide group (s) of the conjugate can undergo a hydrolysis reaction.

  The rate of thio-substituted succinimide hydrolysis can be manipulated by adjusting the reaction conditions following drug-linker conjugation to the ligand, for example by adjusting the pH or temperature. In some embodiments of the invention, all, substantially all, or at least 50%, 60%, 70%, 80%, 85%, 90%, or even 95% of the thio-substituted succinimide is Without hydrolysis, ie the hydrolysis reaction is carried out under the same reaction conditions as the conjugation reaction. In some embodiments, all, substantially all, or at least 50%, 60%, 70%, 80%, 85%, 90% or even 95% of the thio-substituted succinimide is from 20 minutes following conjugation. It is hydrolyzed in 4 hours, preferably 20 minutes to 2 hours following conjugation. In an exemplary embodiment, the conjugation conditions are a pH of about 7.4 and a temperature of about 22 ° C.

In some embodiments, a method of preparing a ligand-drug conjugate comprises providing a drug-linker or linker unit; conjugating said drug-linker or linker unit to a sulfhydryl group of a ligand and hydrolyzing. Forming a ligand-drug conjugate comprising unmodified thio-substituted succinimide; and subjecting the unhydrolyzed thio-substituted succinimide to a hydrolysis reaction, wherein all, substantially all, or at least 50% of the succinimide , 60%, 70%, 80% or even 85% are hydrolyzed in 10 minutes to 4 hours following conjugation. In some embodiments, all, substantially all, or at least 50%, 60%, 70%, 80%, 85%, 90%, or even 95% of the succinimide is up to 10 minutes following conjugation, Hydrolyzed up to 20 minutes, up to 40 minutes, up to 60 minutes, up to 90 minutes or up to 120 minutes. In some embodiments, the hydrolysis reaction occurs under the same reaction conditions as the conjugation reaction. In an exemplary embodiment, the conjugation conditions are a pH of about 7.4 and a temperature of about 22 ° C.
Ligand-Drug Conjugate Assembly The ligand-drug conjugates of the invention can be assembled according to the general scheme outlined in FIG.

Summary Unless otherwise noted, materials were obtained from commercial suppliers in the highest purity grade available and used without further purification. Anhydrous DMF and CH ₂ Cl ₂ were purchased from Aldrich. Fmoc-Dolaproine-OH is available from Albany Molecular Research, Inc. (Albany, NY) custom synthesized. Dolavaline-Val-Dil-OH was prepared as described elsewhere. Fmoc-Dpr (ivDde) -OH and 2-chlorotrityl chloride resin (200-300 mesh, 1% DVB, substitution 1 mmol / gram) were purchased from Novabiochem. Solid phase synthesis was carried out in a plastic syringe (National Scientific Company) fitted with a filter cut out from a middle grade porous sheet (Scienceware). A Burrell wrist action® shaker (Burrell Scientific, Pittsburg, PA) was used for stirring. Unless otherwise stated, all reported solid phase yields are based on the initial substitution level of the resin and constitute the mass balance of isolated pure material.

  Preparative HPLC purification was performed on a Varian instrument equipped with a C12 Phenomenex Synergy MAX-RP 4μ reverse phase column, 250 × 10 mm, eluting with 0.05% TFA in a water-acetonitrile gradient.

  Mass spectral data were obtained on a XEVO TOF MS interfaced with a Waters 2795 HPLC equipped with a C12 Phenomenex Synergi 2.0 × 150 mm, 4 μm, 80Å reverse phase column. The eluent consisted of a 5% to 95% linear gradient of acetonitrile in 0.1% aqueous formic acid over 10 minutes at a flow rate of 0.4 mL / min followed by 95% acetonitrile at a uniform concentration for 5 minutes.

  The humanized h1F6 antibody specifically binds to the human CD70 antigen (Cancer Res, 2006, 66 (4), 2328; US Pat. No. 8,067,546). The humanized hBU12 antibody specifically binds to the human CD19 antigen (Blood, 2009, 113 (18), 4362; US Pat. No. 7,968,687). Human renal cell carcinoma cell lines 786-O and Caki-1 expressing human CD70, as well as human transformed follicular lymphoma DOHH2 cells expressing human CD19, are available from the American Cultured Cell Line Conservation Organization (ATCC; Manassas, Purchased from Virginia). All cell lines were grown according to supplier recommendations and checked for mycoplasma contamination as prescribed.

Abbreviation: DPR means diaminopropionic acid; ivDde is 1- (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) -3-methylbutyl-.
Example 1
General procedure for synthesis

Maleimide -Dpr (Boc) -OH synthetic N β _-Boc-L-2,3- diaminopropionic acid (1 mmol) and maleic acid (98 mg, 1 mmol) and acetic acid (1 mL) in a round bottom flask 50ml Dissolved and the solution was stirred at room temperature for 3 hours. The solution was then concentrated under reduced pressure to an oil. The maleic acid intermediate was precipitated by adding about 10 mL of CH ₂ Cl ₂ / hexane (1/1, v / v) and the precipitate was collected by vacuum filtration. This material was then suspended in toluene (9 mL) followed by the addition of DMA (0.3 mL) and triethylamine (0.42 mL, 3 mmol). The mixture was stirred at 40-60 ° C. under N ₂ until all material was dissolved. The flask was then equipped with a condenser and the solution was heated to 120 ° C. and refluxed on a molecular sieve for 4 hours. The reaction mixture was filtered through a sintered glass funnel and nearly concentrated to dryness under reduced pressure. The residue was dissolved in ethyl acetate (10 mL), transferred to a separatory funnel and washed with 10% citric acid in water (2 × 10 mL) and brine (2 × 10 mL). The organic layer was dried over magnesium sulfate, concentrated under reduced pressure, and dried overnight under high vacuum to give the product as a white powder in 72% yield. ¹ H NMR (DMSO): δ 1.29 (s, 9H), 3.41 (m, 1H), 3.52 (m, 1H) 4.57 (dd, 1H). 6.97 (t, 1H), 7.07 (s, 2H). LCMS (ESI) calculated for (M + Na) ^<+ >307.09; found, m / z 307.17.

General procedure for auristatin-AA ₁ -MDpr synthesis

FIG. 1 illustrates an exemplary synthesis of auristatin- (AA ₂ ) -AA ₁ -MDpr drug-linker.

Resin load. A 20 mL solid phase reaction vessel (plastic syringe with PET frit) is charged with 1 g of 2-chlorotrityl chloride resin (1 mmol based on manufacturer's indication) followed by Fmoc-Dpr (ivDde) -OH or Fmoc-Lys ( ivDde) -OH (1.5mmol, 1.5 eq) and DIEA (1 mmol, 1 dry _{CH 2 Cl 2 / DMF (1} /1, v / v eq), 10 mL) solution was added. The bath was shaken for 5 minutes and then additional DIEA (1.5 mmol, 1.5 eq) was added. The mixture was shaken for an additional 2 hours at RT. Methanol (2.5 mL) was added to quench unreacted sites. After 30 minutes, the resin was washed with DMF (5 × 10 mL), CH ₂ Cl ₂ (5 × 10 mL), ethyl ether (5 × 10 mL) and dried in vacuo.

The loading was determined by treating a small amount of resin (2-4 mg) with 20% piperidine / DMF (2 mL) for 2 hours in a volumetric flask (10 or 20 mL). The volume was adjusted with DMF. Absorption at 301 nm was measured. The amount of load was calculated by the following equation.
Load (mmol / g) = (Flask volume × A ₃₀₁ ) / (7800 × mg) × 1000
The average loading was about 0.6 mmol / g.

Fmoc removal step. The resin containing the Fmoc protected peptide was treated with 20% piperidine in DMF (10 mL per gram of resin) for 2 hours at room temperature. The resin was then washed with DMF (5 × 1 mL per gram of resin), CH ₂ Cl ₂ (5 × 1 mL per gram of resin), ethyl ether (5 × 1 mL per gram of resin) and dried in vacuo. .

Coupling step. To a resin (1 equivalent) containing the deprotected N-terminal amino acid (AA) was added Fmoc-AA-OH (2 equivalents), HATU (2 equivalents), and DIEA (4 equivalents) of DMF (1 mL per gram of resin). ) The solution was added. The reaction vessel was stirred for 3-4 hours. The resin was then washed with DMF (5 × 1 mL per gram of resin), CH ₂ Cl ₂ (5 × 1 mL per gram of resin), ethyl ether (5 × 1 mL per gram of resin) and dried in vacuo. . Completion of the reaction was confirmed by negative Kaiser test as needed.

  Coupling of N-terminal Dravaline-Val-Dil-OH was performed in the same manner.

ivDde deprotection and coupling of MDpr (Boc) -OH. After coupling of Dravalin-Val-Dil-OH tripeptide, the resin was treated with 2% hydrazine / DMF (1 mL per gram of resin) for 2 hours at RT. The resin was then washed with DMF (5 × 1 mL per gram of resin), CH ₂ Cl ₂ (5 × 1 mL per gram of resin), ethyl ether (5 × 1 mL per gram of resin) and dried in vacuo. . A solution of Fmoc-MDpr (Boc) -OH (2 eq), HATU (2 eq), and DIEA (4 eq) in DMF (1 mL per gram of resin) is added to the resin and the mixture is allowed to reach room temperature (RT) for 3 hours. Shake. Completion of the reaction was confirmed by a negative Kaiser test. The resin was washed with DMF (5 × 1 mL per gram of resin), CH ₂ Cl ₂ (5 × 1 mL per gram of resin), ethyl ether (5 × 1 mL per gram of resin) and dried in vacuo.

Cutting and deprotection from resin. The peptide-containing resin was treated with 20% TFA / CH ₂ Cl ₂ (2 mL per gram of resin) for 10 minutes at room temperature and the solution was collected in a round bottom flask. The resin was washed with 20% TFA / CH ₂ Cl ₂ (2 × 0.5 mL per gram of resin). The pooled solution was left at RT for 3 hours. Completion was confirmed by LC-MS after deprotection. Volatiles were removed on Rotavap under reduced pressure and the final product was purified by reverse phase preparative HPLC. All drug-linkers were obtained in> 95% purity by reverse phase HPLC at 215 nm.

  Drug-linker with MA maleimide was prepared in a similar manner as described above using α-maleimidoacetic acid-NHS (Molecular Biosciences, Boulder CO) instead of MDpr (Boc) -OH.

A drug-linker with an ethylenediamine (EDA) stretcher was prepared by a procedure similar to that previously reported (Bioconjugate Chem. 2008, 19, 1960-1963).
(Example 2)
Drug linker The drug linker was synthesized as described above. The general formula was as follows:
(Note that in this equation, the designations of AA ₂ and AA ₁ are reversed. R corresponds to R ^12. )

Table 1 below summarizes the synthesis and characterization of various drug linkers. In the table, the first column (left) refers to the compound number. The second column (left) refers to the amino acid at the C-terminus of auristatin. The third, fourth and fifth columns refer to the components of the linker. In the third column, the amino acid component of the linker is identified. In the fourth column, additional amino acid and / or non-amino acid components of the linker are identified. In column 5, the composition of the maleimide portion of the linker is identified. The sixth column refers to the drug-linker yield. Columns 7 and 8 refer to the calculated and observed masses of the drug-linker as determined by mass spectrometry. The last column (right) refers to the HIC retention time of the ADC containing the drug linker as 8 loads (generally determined as described in Example 3).
Abbreviations:
Ala refers to L-alanine, Asn refers to asparagine, Asp refers to L-aspartate, Gln refers to L-glutamine, Glu refers to L-glutamate, Ile refers to L-isoleucine Leu refers to L-leucine, Lys refers to L-lysine, Phe refers to L-phenylalanine, phosphoThr refers to L-phosphothreonine, Thr refers to L-threonine, hSer is L-hydroxyserine:
ValOH is L-hydroxyvaline:
Pyrazole is
Triazole is
And Fur is
MA refers to maleimidoacetyl, Dpr refers to diaminopropionic acid, MDpr refers to maleimidodiaminopropionic acid, EDA refers to ethylenediamine, mc-MMAF refers to the linker maleimidocaproyl MMAF, mc-vc-PABC-MMAF refers to maleimidocaproyl-valine-citrulline-p-aminobenzyl-carbamoyl MMAF, mc-vc-PABC-MMAF refers to maleimidocaproyl-valine-citrulline-p-aminobenzyl-carbamoyl Refers to MMAE.
(Example 3)
Antibody drug conjugate

Preparation of antibody-drug conjugate. Illustration for h1F6 ADC.

  H1F6 antibody-drug conjugate (ADC) with 8 drugs per antibody was prepared by complete reduction of the antibody followed by reaction with the desired drug-linker. Antibody (10 mg / mL) was added at 10 molar equivalents of tris (2-carboxyethyl) phosphine in phosphate buffered saline (PBS) pH 7.4 (Invitrogen, Carlsbad, Calif.) With 1 mM diethylenetriaminepentaacetic acid (DTPA). (TCEP) was added, followed by complete reduction by incubation at 37 ° C. for about 1 hour. Excess TCEP was removed by repeating the 10-fold dilution in PBS and antibody concentration twice using a 30 KD MWCO spin filter (EMD Millipore, Billerica, Mass.). Complete reduction of the antibody is confirmed by reverse phase HPLC analysis, and the light and heavy chains are completely separated from the non-reducing antibody. Drug-linker (10 eq) was then added from a stock solution prepared in DMSO (10 mM). The reaction was left at room temperature for approximately 2 hours to allow conjugation and subsequent thiosuccinimide ring hydrolysis (MDpr). The reaction mixture was purified using a PD-10 desalting column (GE Healthcare, Piscataway, NJ) and buffer exchanged into PBS. The drug / Ab ratio of the final product was estimated by PLRP-MS analysis, which ranged from 7.8 to 8.0 drugs / Ab. In addition, each ADC was analyzed by size exclusion chromatography and the HMW species ranged from 0.5 to 2.0%.

Hydrophobic interaction chromatography

  Analysis of ADC was performed using hydrophobic interaction chromatography (HIC). HIC is performed by performing a linear gradient from 0-100% mobile phase B (MPB), mobile phase A (MPA) consists of 1.5 M ammonium sulfate, 25 mM potassium phosphate (pH 7.0), MPB consists of 75% 25 mM potassium phosphate (pH 7.0), 25% isopropanol. Separation was achieved using a Tosoh t-butyl column (TSK-Gelbutyl-NPR, 4.6 × 35 mm, PN: 14947) heated to 25 ° C. The test substance was prepared by diluting 70 μg of ADC in MPA so that the total salt concentration was equal to or higher than 1.0 M ammonium sulfate in a total volume of 100 μL. Samples were injected at 90 μL and eluted using a 12 minute gradient. Monitoring at λ = 280 nm. ADCs with greater hydrophobicity or more drugs per molecule will elute at a later retention time.

  4 and 5 show the results of HIC analysis of various 8-loaded ADCs compared to the parent, unconjugated antibody (h1F6). The ADC was prepared as described above. The results generally indicate that increasing the auristatin hydrophilicity in combination with a hydrophilic linker reduces the apparent hydrophobicity of the conjugate.

Table 2 below summarizes the composition of the various drug linkers in Table 1 and the analysis of the 8 loaded antibody drug conjugates thus obtained with antibody h1F6. HIC retention time (HIC RT) was determined as described above. H1F6 ADC containing drug linkers MC-vc-PABC-MMAE, MC-vc-PABC-MMAF and MC-MMAF was used as a control.
Example 4
In vitro activity assay

In vitro cytotoxicity assays were performed as previously described generally (see above, activity assay). Briefly, log phase cultures of cells were collected and cells were seeded according to given conditions at a seeding density ranging from 500 to 10,000 cells / well. After 24 hours of incubation to allow surface protein reconstitution, serial dilutions of the test conjugate were added and the cultures were incubated for an additional 4 days. Assessment of cell growth and dye reduction resulting in IC ₅₀ values were performed using the Alamar Blue (Biosource International, Camarillo, Calif.) Dye reduction assay. Briefly, a 40% solution of Alamar Blue (wt / vol) was freshly prepared in complete media immediately prior to adding the culture. After 92 hours of drug exposure, Alamar Blue solution was added to the cells to make up a 10% culture volume. Cells were incubated for 4 hours and dye reduction was measured with a Fusion HT fluorescent plate reader (Packard Instruments, Meriden, CT).

786-O kidney and Caki-1 clear cell renal cancer cell lines were used. These cell lines expressed approximately 190,000 and 135,000 human CD70 molecules, respectively, per cell. The drug linkers attached to the h1F6 antibody are listed in Table 1 and Table 2. Referring to Tables 3A-C below, h1F6 ADC has an average drug loading of 8 drugs / antibodies unless otherwise indicated. The tested hydrophilic h1F6 ADC showed an activity (IC ₅₀ value) comparable or better than the control, h1F6-mcMMAF (1269) in these studies.
(Example 5)
Pharmacokinetic study

  Antibodies and ADC radiolabels

  Pharmacokinetic (PK) experiments were performed using radiolabeled antibodies or ADC. The PK test substance was radiolabeled using the following procedure. A solution of antibody or ADC in 500 mM potassium phosphate (pH 8.0) and 500 mM sodium chloride was added to 1 mg antibody or 55 μCi N-succinimidyl propionate, [propionate-2,3-3H. ]-(Moravek Biochemicals, catalog number: MT919, 80 Ci / mmol, 1 mCi / mL, 9: 1 hexane: ethyl acetate solution) was added. The resulting mixture was vortexed and allowed to stand at room temperature for 2 hours. The mixture was centrifuged at 4,000 × g for 5 minutes, the lower aqueous layer was removed and divided into Amicon Ultra-15 Centrifugal Filter Units (Millipore, Catalog Number: UFC903024, 30 kDa MWCO). Unconjugated radioactivity was removed by 4 rounds of dilution and centrifugation at 4,000 × g. The product thus obtained was filtered through sterile 0.22 μm Ultrafree-MC Centrifugal Filter Units (Millipore, catalog number: UFC30GV0S) and the final antibody or ADC concentration was measured spectrophotometrically. The specific activity (μCi / mg) of each product was determined by liquid scintillation counting.

  Pharmacokinetic study

  The pharmacokinetic properties of unconjugated antibody and its various ADCs (8 drug loadings) were investigated in several rodent models. In each experiment, 3 mg of radiolabeled antibody or ADC per kg animal body weight was injected through the tail vein. Each test substance was administered once in 3 animals of the same type. Blood was collected into K2EDTA tubes via the saphenous vein or by cardiac puncture for end-stage bleeding at various time points. Plasma was isolated by centrifugation at 10,000 xg for 10 minutes. A 10 μL sample of plasma from each time point was added to 4 mL of Ecoscint-A liquid scintillation cocktail (National Diagnostics) and total radioactivity was measured by liquid scintillation counting. The resulting decay per minute value was converted to μCi and the specific activity of the radiolabeled test substance was used to calculate the concentration of antibody or ADC remaining in the plasma at each time point.

Referring to FIG. 2, the pharmacokinetic properties of h1F6 and its two hydrophilic ADCs were compared with those of three control ADCs. Hydrophilic ADCs are h1F6-4 (8-loaded (auristatin-T) -Glu-Dpr-MA) ₈ -h1F6) and h1F6-11 ((auristatin F) -Ile-EDA-MDpr) ₈ -h1F6. )Met. The results show that the hydrophilic ADC showed improved pharmacokinetic stability over this mouse study. Hydrophilic auristatin with auristatin-T showed stability similar to that of unconjugated antibody. Although the hydrophilic design of ADC h1F6-11 showed improved pK stability compared to the control, these two contain the same monomethyl form of auristatin (auristatin F vs. monomethyl auristatin F) .

  Referring to FIG. 3, the pharmacokinetic properties of another monoclonal antibody hydrophilic conjugate were compared to those of a control conjugate, mAb-mcMMAF. All of the ADCs had an average drug load of 8. Each of the hydrophilic ADCs showed improved pharmacokinetic stability compared to the control ADC.

The two hydrophilic ADCs were compared to the characteristics of three control ADCs. Hydrophilic ADCs are h1F6-8 (8-loaded (auristatin-thiazole) -Glu-Lys-MDpr) ₈ -h1F6) and h1F6-11 ((auristatin F) -Ile-EDA-MDpr) ₈ -h1F6. )Met. The results show that the hydrophilic ADC showed improved pharmacokinetic stability over this mouse study. In particular, the hydrophilic design of h1F6-11 showed improved stability compared to the control, but these two contain the same monomethyl form of auristatin (auristatin F vs. monomethyl auristatin F). .
(Example 6)
In vivo treatment experiments

786-O cells were obtained from the American Cultured Cell Line Conservation Agency (ATCC, Manassas, Va.) And propagated in culture conditions recommended by ATCC. To establish 786-O tumors, 5 × 10 ⁶ cells were transplanted into the right flank of athymic nu / nu female donor mice (Harlan, Indianapolis, IN). When donor tumors were approximately 500 mm ³ , mice were euthanized, tumors were aseptically removed, and approximately 0.5 × 0.5 mm fragments were sterilized for transplantation into nu / nu mice. Filled in 13 gauge trocar. When tumors reached approximately 100 mm ³ , mice were randomly assigned to treatment groups.

To establish DOHH2 tumors, 5 × 10 ⁶ cells were injected into C. B. -17 SCID mice (Harlan, Indianapolis, IN) were implanted into the right flank. Mice were randomly assigned to treatment groups when tumors were approximately approximately 100 mm ³ .

Experimental groups were treated with compounds by intraperitoneal injection at the indicated doses and schedules or left untreated instead. Tumors were measured periodically and volume was calculated using the formula V = ((L × W ² ) / 2). Animals were euthanized when tumors reached a volume of 1000 mm ³ or when the end of the study first came.

  The time at which tumors quadrupled was selected as the time to endpoint (TTE), which was determined using nonlinear regression analysis for exponential growth of each individual tumor growth data set from each experimental animal. did. The median time for tumor to quadruple was calculated based on the tumor volume at the start of treatment. Animals that did not reach the endpoint were assigned a TTE value equal to the last day of the study.

  Statistical analysis was performed using Prism (GraphPad) software for Windows®. TTE log rank test was used to analyze the significant difference between the two groups, the difference is considered significant at 0.01 ≦ P ≦ 0.05, and highly significant at P ≦ 0.01 Considered.

  Referring to FIG. 6, the activity of 4 and 8 loaded ADCs (4d / Ab and 8d / Ab, respectively) was tested in a single dose mouse xenograft study. First, referring to the control, h1F6-mc-vc-PABC-MMAF, the 4-loaded ADC gave better activity than the 8-loaded ADC. In contrast, both hydrophilic h1F6-6 (auristatin T-Glu-Dpr-MDPr) and h1F6-13 (auristatin thiazole-Glu-EDA-MDPr) 8-loaded ADCs were 4-loaded counters. Greater activity than part.

  Referring to FIG. 7, the activity of different 4- and 8-loaded ADCs was tested in a single dose mouse xenograft study. Again in this model, the 8-loaded hydrophilic ADC of hBU12-6 (Auristatin T-Glu-Dpr-MDPr) showed greater activity than its 4 loaded counterpart.

  Referring to FIG. 8, the activity of various 4- and 8-loaded ADCs was tested in a single dose mouse xenograft study. In this model, both h1F6-12 (Auristatin T-Ile-EDA-MDPr) and h1F6-5 (Auristatin F-Glu-Dpr-MDPr) 8-loaded ADCs had 4 loaded counterparts. Greater activity was shown. Eight loaded ADC h1F6-11 (Auristatin F-Ile-EDA-MDPr) showed the opposite trend.

  Referring to FIG. 9, the activity of various 4- and 8-loaded ADCs was tested in a single dose mouse xenograft study. In this model, 8 loaded ADCs of h1F6-17, h1F6-20, h1F6-24 and h1F6-29 showed greater activity than the 4 loaded counterparts.

Claims (27)

formula
A hydrophilic drug-ligand conjugate compound, or a pharmaceutically acceptable salt or solvate thereof, comprising:
Where
L is a ligand that specifically binds to the target;
L ^A is a ligand binding component,
L ^H is a hydrophilic linker that is optionally branched, and each branch of L ^H has the formula -AA ₁ -R ^L1 -R ^L2 -R ^L3-
Have
Where
AA ₁ is a hydrophilic amino acid that forms a cleavable bond with _DE to which it is attached;
R ^L1 is optional, and when R ^L1 is absent exists vital R ^L2 and R ^L3, alkylene substituted with a hydrophilic amino acid or optionally may share L ^A and atoms,
R ^L2 is optional, and when R ^L2 is not present there vital R ^L3, are selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
R ^L3 is optional, and when R ^L3 is present, is selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
The subscript p is an integer from about 4 to about 20,
The subscript p ′ is an integer from 1 to 4,
Each _DE is a formula
Or a pharmaceutically acceptable salt or solvate thereof,
Where
Each of R ¹ and R ² is independently selected from the group consisting of hydrogen (H) and optionally substituted —C ₁ -C ₈ alkyl, provided that both R ³ and R ^{3 ′} are not H Except in cases, R ¹ and R ² are not both H,
R ³ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
R ^{3 ′} is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
At least one of R ³ and R ^{3 ′} is not H,
R ⁴ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
R ⁵ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
Or R ⁴ and R ⁵ together form a carbocyclic ring and the formula — (CR ^a R ^b ) _n — (wherein R ^a and R ^b are H and optionally substituted —C Independently selected from the group consisting of _{1 to} C ₈ alkyl, and n is selected from the group consisting of 2, 3, 4, 5 and 6.
R ⁶ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
R ⁷ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
Each R ⁸ is independently from the group consisting of H, —OH, optionally substituted —C ₁ -C ₈ alkyl, and optionally substituted —O— (C ₁ -C ₈ alkyl). Selected
R ¹² is threonine, serine, asparagine, aspartic acid, glutamine, glutamic acid, homoserine, hydroxyvaline, furylalanine, threonine (PO ₃ H ₂ ), pyrazolylalanine, triazolylalanine and thiazolylanine, or a pharmaceutical thereof Selected from the group of side chains consisting of salts or solvates acceptable to
Left and right lines L ^H, respectively, indicate a covalent bond to the _{D E} units and ^{L A,}
The drug-ligand conjugate is a hydrophilic drug-ligand conjugate compound, or a pharmaceutically acceptable salt or solvate thereof, having a hydrophilicity index of less than 2. formula
A drug-linker compound having the formula: or a pharmaceutically acceptable salt or solvate thereof,
Where
L ^A is a ligand binding component,
L ^H represents the formula -AA ₁ -R ^L1 -R ^L2 -R ^L3-
A hydrophilic linker that is optionally branched with
Where
AA ₁ is a hydrophilic amino acid that forms a cleavable bond with the _DE unit to which it is attached;
R ^L1 is optional, and when R ^L3 and R ^L3 are absent, are selected from alkylene substituted with hydrophilic amino acids, or optionally may share L ^A and atoms,
R ^L2 is optional, and when R ^L2 is not present there vital R ^L3, are selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
R ^L3 is optional, and when R ^L3 is present, is selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
L ^A is a ligand binding component,
The subscript p ′ is an integer from 1 to 4,
Each _DE is a formula
Auristatin (Aur), or a pharmaceutically acceptable salt or solvate thereof,
Where
Each of R ¹ and R ² is independently selected from the group consisting of hydrogen (H) and optionally substituted —C ₁ -C ₈ alkyl, provided that both R ³ and R ^{3 ′} are not H Except in cases, R ¹ and R ² are not both H,
R ³ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
R ^{3 ′} is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
At least one of R ³ and R ^{3 ′} is not H,
R ⁴ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
R ⁵ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
Or R ⁴ and R ⁵ together form a carbocyclic ring and the formula — (CR ^a R ^b ) _n — (wherein R ^a and R ^b are H and optionally substituted —C Independently selected from the group consisting of _{1 to} C ₈ alkyl, and n is selected from the group consisting of 2, 3, 4, 5 and 6.
R ⁶ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
R ⁷ is selected from the group consisting of H and optionally substituted —C ₁ -C ₈ alkyl;
Each R ⁸ is independently from the group consisting of H, —OH, optionally substituted —C ₁ -C ₈ alkyl, and optionally substituted —O— (C ₁ -C ₈ alkyl). Selected
R ¹² is threonine, serine, asparagine, aspartic acid, glutamine, glutamic acid, homoserine, hydroxyvaline, furylalanine, threonine (PO ₃ H ₂ ), pyrazolylalanine, triazolylalanine and thiazolylanine, or a pharmaceutical thereof Selected from the group of side chains consisting of salts or solvates acceptable to
L left and right lines of ^H, respectively, indicates the covalent attachment to D _E units and L ^A, Drug - Linker Compound or a pharmaceutically acceptable salt or solvate thereof. Wherein L ^H comprises a modified peptide and at least one of R ^L1 , R ^L2 and R ^L3 is an amino acid covalently linked to an adjacent group by a reactive group on its side chain, Or a pharmaceutically acceptable salt or solvate thereof. A compound according to any preceding claim, or a pharmaceutically acceptable salt or solvate thereof, wherein R ¹² is a side chain of L-threonine.   5. The drug-ligand conjugate compound according to any one of claims 1 and 3 to 4, or a pharmaceutically acceptable salt or solvate thereof, wherein p of the drug-ligand conjugate is at least 8.   6. A compound according to claim 5, or a pharmaceutically acceptable salt or solvate thereof, wherein p of the drug-ligand conjugate is at least 10.   6. The compound according to claim 5, or a pharmaceutically acceptable salt or solvate thereof, wherein p of the drug-ligand conjugate is at least 16. ^A compound according to any preceding claim, or a pharmaceutically acceptable salt or solvate thereof, wherein LA is succinimide or hydrolyzed succinimide. Formula _{^{(a) (L H) p}} '-L A
Or a pharmaceutically acceptable salt or solvate thereof, wherein
L ^H is a hydrophilic linker that is optionally branched, and each branch of L ^H has the formula -AA ₁ -R ^L1 -R ^L2 -R ^L3-
Have
L ^A is a ligand binding component,
p ′ is an integer of 1 to 4,
Left and right lines L ^H, respectively, indicating the binding sites for _{D E} units and ^{L A),} or formula ^{(b) ([L H]} p '-L A) p -L
Or a pharmaceutically acceptable salt or solvate thereof, wherein
L is a ligand that specifically binds to the target;
L ^H is a hydrophilic linker that is optionally branched, and each branch of L ^H has the formula -AA ₁ -R ^L1 -R ^L2 -R ^L3-
Have
L ^A is a ligand binding component,
The subscript p is an integer of about 4 to 20,
The subscript p ′ is an integer from 1 to 4,
Left and right lines L ^H, respectively, a indicates the binding site on the D _E units and L ^A),
Here, in (a) or (b),
AA ₁ is a hydrophilic amino acid that can form a cleavable bond with the _DE unit when it is attached;
When R ^L2 and the R ^L3 is absent, R ^L1 is alkylene substituted with a hydrophilic amino acid or optionally may share L ^A and atoms,
R ^L2 is optional, and when R ^L2 is not present there vital R ^L3, are selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atoms,
R ^L3 is optional, and when R ^L3 is present, is selected from alkylene substituted with hydrophilic amino acids and optionally may share L ^A and atom, compound, or a pharmaceutically acceptable Salt or solvate. (A) AA ₁ is a hydrophilic amino acid selected from the group consisting of glycine and L-form of aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine;
(B) When R ^L1 is present, it is
Aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine L-type or D-type; —NH—CH (R ^a ) —CO—; and —NH—CH (COOH) —R ^b - (wherein, ^{R a} _{is, -CH 2 NH 2, -CH 2} CH 2 NH 2, -CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 is selected from _{OH, -CH 2 CH 2 CH 2 CO 2} H, -CH 2 CH 2 CH 2 CO 2 H and _{-CH 2 CH 2 CH 2 CH 2} CO 2 H, R b _is, -CH 2 NH-, _{-CH 2 CH 2 NH -, -} CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CO -, - CH 2 CH 2 CH 2 CO—, —CH ₂ CH ₂ CH ₂ CO— and —CH ₂ CH ₂ CH ₂ CH ₂ CO—); and —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl), -NH ₂ or -NH (C ₁ -C ₃ alkyl) selected from the group consisting of C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents And
(C) when R ^L2 is present,
Aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine L-type or D-type; —NH—CH (R ^a ) —CO—; and —NH—CH (COOH) —R ^b - (wherein, ^{R a} _{is, -CH 2 NH 2, -CH 2} CH 2 NH 2, -CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 is selected from _{OH, -CH 2 CH 2 CH 2 CO 2} H, -CH 2 CH 2 CH 2 CO 2 H and _{-CH 2 CH 2 CH 2 CH 2} CO 2 H, R b _is, -CH 2 NH-, _{-CH 2 CH 2 NH -, -} CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CO -, - CH 2 CH 2 CH 2 CO—, —CH ₂ CH ₂ CH ₂ CO— and —CH ₂ CH ₂ CH ₂ CH ₂ CO—); and —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl), -NH ₂ or -NH (C ₁ -C ₃ alkyl) selected from the group consisting of C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents And
(D) When R ^L3 is present, it is
Aspartate, glutamate, asparagine, glutamine, histidine, lysine, arginine, serine and alanine L-type or D-type; —NH—CH (R ^a ) —CO—; and —NH—CH (COOH) —R ^b - (wherein, ^{R a} _{is, -CH 2 NH 2, -CH 2} CH 2 NH 2, -CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 CH 2 CH 2 NH 2, -CH 2 CH 2 is selected from _{OH, -CH 2 CH 2 CH 2 CO 2} H, -CH 2 CH 2 CH 2 CO 2 H and _{-CH 2 CH 2 CH 2 CH 2} CO 2 H, R b _is, -CH 2 NH-, _{-CH 2 CH 2 NH -, -} CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CH 2 CH 2 NH -, - CH 2 CH 2 CO -, - CH 2 CH 2 CH 2 CO—, —CH ₂ CH ₂ CH ₂ CO— and —CH ₂ CH ₂ CH ₂ CH ₂ CO—); and —NH—, —C (O) —, —COOH, —N (C ₁ -C ₃ alkyl), -NH ₂ or -NH (C ₁ -C ₃ alkyl) selected from the group consisting of C ₁ -C ₆ alkylene optionally substituted with 1 to 4 substituents To be
A compound according to any of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof. (A) AA ₁ is present and R ^L1 , R ^L2 and R ^L3 are absent,
(B) AA ₁ is present, R ^L1 is present, R ^L2 and R ^L3 are absent,
(C) AA ₁ is present, R ^L1 is present, R ^L2 is present, and R ^L3 is absent,
(D) AA ₁ is present, R ^L1 is present, R ^L2 is present, R ^L3 is present,
(E) AA ₁ is present and at least one of R ^L1 , R ^L2 and R ^L3 is present and is optionally substituted alkylene,
(F) AA ₁ is glutamate and at least one of R ^L1 , R ^L2 and R ^L3 is present and optionally substituted alkylene,
(G) AA ₁ is glutamate, R ^L1 is a hydrophilic amino acid, at least one of R ^L2 and R ^L3 is present and optionally substituted alkylene,
(H) AA ₁ and R ^L1 are hydrophilic amino acids, at least one of R ^L2 and R ^L3 is present and optionally substituted alkylene,
(I) AA ₁ is a hydrophilic amino acid and R ^L1 and optionally R ^L2 are optionally substituted alkylene,
(J) AA ₁ is present, at least one of R ^L1 , R ^L2 and R ^L3 is present, and ethylenediamine, —NH—CH (COOH) —CH ₂ —NH— and —CO—CH (CH ₂ NH ₂ )-Is an optionally substituted alkylene selected from the group consisting of
(K) AA ₁ is glutamate, R ^L1 is a hydrophilic amino acid, at least one of R ^L2 and R ^L3 is present, and ethylenediamine, —NH—CH (COOH) —CH ₂ —NH— and _{-CO-CH (CH 2 NH 2} ) - or is alkylene optionally substituted with selected from the group consisting of,
(L) AA ₁ and R ^L1 are hydrophilic amino acids, at least one of R ^L2 and R ^L3 is present, and ethylenediamine, —NH—CH (COOH) —CH ₂ —NH— and —CO—CH ( CH ₂ NH ₂ ) — is an optionally substituted alkylene, or (m) AA ₁ is a hydrophilic amino acid, and R ^L1 and optionally R ^L2 are ethylenediamine _{, -NH-CH (COOH) -CH} 2 -NH- and _{-CO-CH (CH 2 NH 2} ) - alkylene which is optionally substituted selected from the group consisting of,
A compound according to any of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof. -L ^H -is the formula
Where
R ²¹ is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, —CH ₂ CH ₂ OH, —CH ₂ CO ₂ H, —CH ₂ CH ₂ CO ₂ H, —CH ₂ CH Selected from the group consisting of ₂ CH ₂ CO ₂ H, and —CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H;
R ²² is selected from the group consisting of —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH;
The wavy lines on the left and right indicate, respectively, a compound according to any of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, indicating a bond to D _E , H or a protecting group, and L ^A , respectively. object. -L ^H -is the formula
Wherein R ²² is selected from —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, and —CH ₂ CH ₂ OH, and the left and right wavy lines are respectively ^A compound according to any of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, which exhibits a bond to D _E , H or a protecting group, and LA. -L ^H-
Having a formula selected from the group consisting of:
Wherein the left and right wavy lines respectively indicate a bond to D _E , H or a protecting group, and L ^A , or a pharmaceutically acceptable salt thereof. Or a solvate. (A) L ^A -L ^H of the drug-linker compound has the formula
Have
In the formula, each R ³¹ is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, —CH ₂ CH ₂ OH, —CH ₂ CO ₂ H, —CH ₂ CH ₂ CO ₂ H, selected _{-CH 2 CH 2 CH 2 CO 2} H, and independently from the group consisting of _{-CH 2 CH 2 CH 2 CH 2} CO 2 H, each of the bars may indicate the binding sites for _{D E} units, or (B) L ^A -L ^H of (a), wherein the maleimide group is covalently bonded to the thiol group of the ligand unit;
A compound according to any of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof. L ^A -L ^H of the drug-linker compound has the formula
Have
A compound according to any preceding claim, wherein each of said bars represents a binding site for a _DE unit, and wherein said maleimide group is optionally covalently bound to a thiol group of a ligand unit, or a pharmaceutical thereof Acceptable salt or solvate. The drug - ^-L A -L ^H ligand conjugate compound has the formula
Where
R ²¹ is —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH, —CH ₂ CH ₂ OH, —CH ₂ CO ₂ H, —CH ₂ CH ₂ CO ₂ H, —CH ₂ CH Selected from the group consisting of ₂ CH ₂ CO ₂ H, and —CH ₂ CH ₂ CH ₂ CH ₂ CO ₂ H;
R ²² is selected from the group consisting of —CH ₂ NH ₂ , —CH ₂ CH ₂ NH ₂ , —CH ₂ OH;
The compound according to any of the preceding claims, wherein the left and right wavy lines indicate binding to _DE and said ligand unit (L), respectively, and said sulfur atom is derived from said ligand unit; Or a pharmaceutically acceptable salt or solvate thereof.   A compound according to any of the preceding claims, wherein the ligand unit (L) of the drug-ligand conjugate or the ligand-linker compound is a protein, polypeptide or peptide.   A compound according to any preceding claim, or a pharmaceutically acceptable salt or solvate thereof, wherein said ligand unit (L) of said drug-ligand conjugate or said ligand-linker compound is an antibody. . ^A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, wherein LA is selected from the group consisting of maleimide or maleimide diaminopropionic acid. A hydrophilic drug-ligand conjugate according to claim 1 having the formula selected from: or a pharmaceutically acceptable salt or solvate thereof,
Wherein S is a hydrophilic drug-ligand conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein S is a sulfur atom of the ligand.   A method of treating a patient in need thereof, said method comprising administering to said patient a drug-ligand conjugate compound according to any of the preceding claims, wherein said patient has cancer. A method having an autoimmune disease or infection, wherein the ligand of the drug-ligand conjugate compound specifically binds to a target cell associated with the cancer, autoimmune disease or infection.   24. The method of claim 22, wherein the drug-ligand conjugate compound is administered at a dose of 0.1-10 mg / kg. (A) p is at least 8 and the dose of the drug-ligand conjugate compound administered to the patient is the same or less than the dose of the bi-loaded conjugate with the same drug linker And
(B) p is at least 8 and the dose of the drug-ligand conjugate compound administered to the patient is equal to or less than the dose of the 4-loaded conjugate with the same drug linker And
24. A method according to any of claims 22 to 23, wherein the drug-ligand conjugate compound and the 2- or 4-loaded conjugate are administered on a comparable schedule.   25. A method according to any one of claims 22 to 24, wherein the patient is a human.   An effective amount of a drug-ligand conjugate compound according to any of claims 1 and 2 to 21, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier or additive. Pharmaceutical composition. Optionally,
(A) the drug-ligand conjugate compound is formulated in unit dosage injectable form;
(B) the amount of the drug-ligand conjugate compound administered to the patient ranges from about 0.1 to about 10 mg / kg of the patient's body weight; or (c) the drug-ligand conjugate compound comprises: Administered intravenously,
27. A pharmaceutical composition according to claim 26 for use in a method for the treatment of cancer, autoimmune disease or infection.